ABSTRACT AN AUTOMATED, COMPUTER-CONTROLLED STOPPED-FLOW SPECTROPHOTOMETER FOR FAST REACTION-RATE ANALYSIS BY Phillip Kent Notz A first generation stopped-flow spectrophotometer has been improved, automated and interfaced to a PDP 8/e mini- computer. The stopped-flow mixing system was completely thermostated and equipped to permit the measurement of ab- sorbance, conductance and temperature on the millisecond time scale. Other design changes include a novel mixer, high throughput quartz optics and an optical trigger system. The operation of the stopped-flow mixing system and the collec- tion, analysis, display and storage of data are all done under minicomputer control. On-line display of results via a high speed CRT terminal provides the experimenter with valuable feedback in seconds after the completion of a stopped-flow experiment. The detection system can achieve a resolution of 1 part in 200,000, which allows the measurement of very small changes in absorbance. The overall accuracy of absorbance measurements is limited to about 0.5% due to drift in light source intensity. Temperature data, with a precision of 0.0 qui 510‘ the ize< accx peri thic were riu: and in S COnS. mOly] Phillip K. Notz 0.01°C, can be acquired nearly simultaneously with the ac— quisition of absorbance data. A complete characterization of the automated stopped- flow spectrophotometer is presented. The components of the mixing and spectrophotometric systems were character- ized to determine their individual effects on the system accuracy and precision. As a check on the overall system performance, rate and equilibrium constants for the iron- thiocyanate reaction were determined. Equilibrium constants were determined at different concentrations, both by equilib- rium absorbance measurements and by fitting both the forward and reverse rate constants to kinetics data. The equilibrium constants (for a given concentration of reactants), as de- termined by the two different methods, agreed by better than one percent in all cases. A preliminary study of the proton consumption by Mo(VI) in strongly acid solution shows that the number of protons consumed per molybdenum atom approaches 2% as the acid-to- molybdate ratio is increased to 10. A study of the dependence of the rate of formation of lZ-molybdophosphate (lZ-MPA) on nitric acid concentration is presented. Results indicate inverse first and inverse ninth order dependence on acid concentration and the formae tion of two different products at different acid concen- trations. Finally, the reaction—rate analysis of phosphate and the reaction rate analysis of silicate via their reactions with Mo(VI) are presented. AN AUTOMATED, COMPUTER-CONTROLLED STOPPED-FLOW SPECTROPHOTOMETER FOR FAST REACTION-RATE ANALYSIS BY Phillip Kent Notz A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirement for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1977 Dedicated to Eleanor, Jennifer and Ryan, and Mom and Dad ii L; 5‘ WC . pre an: and the Gal QM on 0r C « AC KNOWLEDGMENTS The author acknowledges the encouragement and guidance of Dr. S. R. Crouch throughout this work. The author expresses gratitude to Dr. C. G. Enke for serving as second reader and for providing helpful sugges- tions and also to Dr. J. L. Dye for meaningful discussions. Dr. Paul Beckwith deserves mention for his initial work in developing the stopped-flow instrument. Jim Holler has been especially valuable both as a friend and as a fellow chemist. His help in completing this work is greatly ap- preciated. Dr. Eric Johnson has provided valuable assist- ance in the development of the computer software. Wai Law and Roy Gall are remembered for their friendship and help in this work. Charlie Patton deserves special mention for the "good vibrations" and for adding color to the lab as only a "running water, slop jar chemist" could. The other members of the author's research group are acknowledged for their friendship, encouragement and helpful discussions. Chuck Hacker and Russ Geyer are acknowledged for their excellent work in the machining of the stopped-flow mixing system. Marty Rabb and Ron Haas deserve mention for their help in the design and construction of electrical components. Mrs. Bernice Wallace deserves recognition for maintaining excellent library facilities. The author is grateful to NSF for research assistant- ships and to Michigan State University for providing iii teaching assistantships. Finally, the author acknowledges his wife, Eleanor, and his parents for their love, understanding and encourage- ment. iv Chapter LIST OF LIST OF CHAPTER A. B. CHAPTER A. CHAPTER A. TABLE OF CONTENTS TABLES . . . . . . . . . . . . . . . FIGURES. . . . . . . . . . . . . . . I - INTRODUCTION . . . . . . . . . . Reaction-Rate Methods of Analysis. . Techniques for Studying Fast Reactions. . . . . . . . . . . . . . 1. Fast Mixing Methods. . . . . . . 2. Relaxation Methods . . . . . . . II - BACKGROUND. . . . . . . . . . . The Stopped-Flow Technique . . . . . 1. Principles of Stopped-Flow MiXing C I I C O O O O O O O C I 2. The Components of a Stopped- Flow Mixing System . . . . . . . 3. Manual Stopped-Flow Systems. . . 4. Automated Stopped-Flow Systems . The Chemistry of lZ-Molybdophosphate l. Molybdenum(VI) in Aqueous Solution . . . . . . . . . . . . 2. The Formation of lZ-Molybdophos- phate O O O O O O O O O O O O I 0 III - THE COMPUTER-CONTROLLED STOPPED-FLOW SPECTROPHOTOMETER The Stopped-Flow Mixing System . . . l. Reagent Delivery and Drive Mechanism. . . . . . . . . . . . 2. Mixer. . . . . . . . . . . . . . 3. Observation Cell . . . . . . . . 4. Stopping Syringe and Trigger . . Page viii 19 26 29 36 36 38 40 41 44 45 48 48 Chapter E. CHAPTER CHAPTER A. CHAPTER CHAPTER A. B. 5. Sequence of Operations . . . . . The Spectrophotometric Detection System . . . . . . . . . . The Thermistor and Thermostating System 0 O I O O O O O O O O O O O 0 Computer Interface . . . . . . . . . Software . . . . . . . . . . . . . . IV - TESTING AND CALIBRATION OF THE AUTOMATED STOPPED-FLOW SPECTROPHOTOMETER . . . . . . . Testing and Calibration of the Flow System. C O I O O O O O O O O 0 Testing and Calibration of the Detection System . . . . . . . . . . Accuracy of Monitoring a Chemical Reaction . . . . . . . . . . . . . . V - STUDY OF THE FORMATION OF lZ-MOLYBDOPHOSPHATE. . . . . . . Proton Consumption by Molybdenum(VI) 1. Characterization of the pH Instrument O O O O O O O O O O O 2. Protons Consumed by Molybdenum(VI) . . . . . . . . . Spectra and Properties of Molybdenyl and lZ-MPA Solutions . . . . . . . . Kinetics of the Formation of 12-MPA in Nitric Acid Solutions . . . . . . VI - THE REACTION-RATE ANALYSIS OF PHOSPHATE AND SILICATE . . . VII - FUTURE PROSPECTS . . . . . . . The Automated Stopped-flow Instrument . . . . . . . . . . . . . Study of the Formation of 12- Molybdophosphate and Related Mo(VI) Compounds . . . . . . . . . . vi Page 51 52 56 58 S9 63 63 67 7O 78 78 78 80 87 92 108 117 117 120 Chapter APPENDIX A - APPENDIX B - APPENDIX C - APPENDIX D - APPENDIX E - BIBLIOGRAPHY INSTRUMENT AND COMPONENT SPECIFICATIONS. . . . . . . . A BRIEF DESCRIPTION OF THE CAPABILITIES OF THE COMPUTER PROGRAMS I O C C O I O O O O I DIALOG FOR PAL8 PROGRAM WHICH OPERATES THE STOPPED-FLOW AND ACQU I RE 8 DATA 0 o o o o o o o PAL8 PROGRAM, PNSF1.PA, WHICH OPERATES THE STOPPED-FLOW AND ACQUIRES SPECTROPHOTOMETRIC DATA. . . . . . . . . . . . . FORTRAN PROGRAM, PNF401.FT, CALCULATES ABSORBANCE AND THE FIRST DERIVATIVE OF ABSORBANCE. . . . . . . . . . vii Page 124 131 135 137 182 189 Table 0m“ 10A 103 11 12 13 LIST OF TABLES Iron-Thiocyanate Concentrations. Iron-Thiocyanate Results Using All 100 Data Points. . . . . . Iron-Thiocyanate Results Using First 20 Data Points . . . . . Iron-Thiocyanate Equilibrium Results. . . . . . . . . . . . Accuracy of the pH Instrument. Mo(VI) in Nitric Acid. . . . . Mo(VI) in Sulfuric Acid. . . . Molar Absorptivity of Mo(VI) . Molar Absorptivity of 12-MPA SOlutions O I O O O O O O O O 0 Initial Rate of Formation of lZ-MPA, Conditions I . . . . . . . . . Initial Rate of Formation of lZ-MPA, Conditions I . . . . . . . . . Initial Rate of Formation of 12-MPA, Conditions II. . . . . . . . . Determination of Rate Constants, Conditions I . . . . . . . . . Determination of Rate Constants, Conditions II. . . . . . . . . viii Page 72 73 74 76 81 83 85 88 93 97 98 100 103 104 Table 14 15 16 Page Reaction-Rate Analysis of Phosphate. . . . . . . . . . . . . . . . llO Reaction-Rate Data: Phosphate Analysis . . . -,- . . . ... . . . . . . 113 Reaction-Rate Data: Silicate Analysis . . . . . . . . . . . . . . ; . 115 ix Figure 10 11 12 LIST OF FIGURES An Automated Stopped—Flow Mixing System with Spectrophotometric Detection. . . . . . . . . . . . . . A Stopped-Flow SpectrOphotometer The Stopped-Flow Mixer . . . . . . . The Stopped-Flow Observation Cell and Quartz Optics . . . . . . Mixing Efficiency. . . . . . . . . . Molar Absorptivity of lZ-MPA . . . Absorbance and the First Derivative of Absorbance, 12-MPA Reaction . . . Dependence of the Rate of Forma- tion of lZ-MPA on [HNO3], Conditions I. . . . . . . . . . . . . . . . . . Dependence of the Rate of Formation of lZ-MPA on [HNO3], Conditions II . Reaction Rate Analysis of Phosphate, Low [Mo(VI)].. . . . . . . . . . . . Analytical curve for Reaction-Rate Analysis of Phosphate. . . . . . . . Analytical curve for the Reaction- Rate Analysis of Silicate. . . . . . Page 11 42 47 49 66 91 96 101 105 111 114 116 tl' in in 1'01 CHAPTER I INTRODUCTION A. Reaction-Rate Methods of Analysis As implied by the name, reaction-rate methods utilize the rate, rather than the stoichiometry, of a reaction to perform a chemical analysis. Several books (1,2) and quite a few review articles (3-10) have been recently written on reaction-rate methods of chemical analysis. Reaction-rate methods of analysis have been developed on a broad scale in recent years. This development is largely due to advances in instrumentation and modern elec— tronics. Until the last decade, the growth of rate methods was inhibited because of the need for more complex analysis procedures and instrumentation than those required for equilib- rium-based methods. However, with the advent of modern integrated circuit electronics and the consequent upsurge in small computer usage, the automation of reaction-rate instruments and reaction—rate data analysis has become routine and relatively inexpensive. Another item which has given great impetus to the development of reaction-rate methods of analysis is the growing realization of the importance of enzymes in bio- logical systems. In many diseases the enzyme level, or enzyme activity, is a critical parameter. Since enzymes f." w: w. q w are biological catalysts, the determination of enzyme activity necessitates reaction-rate procedures. For a general chemical reaction, the rate of disappea- rance of a reactant A with time may be expressed in terms of the concentrations of the chemical species involved in the reaction. -dA _ m n TE- - kl[A] [B] o o 0 Equations relating the rate of disappearance of A or the concentration of A at any time t with the initial con- centration of A, [A]o can be developed for specific cases. In rate methods of analysis conditions are usually chosen such that the reaction is first-order or pseudo-first-order in the species of interest. Thus the following equations would apply: 5A _ a-E - REA] (1) [Alt = [A]o exp(-kt) (2) «159% = kmo an-..) (3) where k is the first-order or pseudo-first-order rate constant and the subscript t indicates the value of the variable at time t. Equation (3) forms the basis for reaction-rate methods m t} fc ma 1i ta Prt of analysis. It indicates that the rate of a first—order reaction at a given time t is related to the initial con- centration of the analyte by a constant, k exp(-kt). Thus the measurement of the reaction-rate at any fixed point in time can form the basis for analysis. In practice the rate must be measured over some finite time interval rather than at a point in time. If this time interval is during the initial portion of the reaction (t << %) the method is termed an initial rate method. In that case the exponential term in Equation (3) is approximately unity. The validity of this approximation has been discussed by Ingle and Crouch (11) and by Crouch (12). In addition, the reaction-rate is a maximum during the initial portion so that sensitivity is optimum there. In comparing reaction-rate methods to equilibrium based methods, the most obvious advantage of the rate method is the shorter measurement time. This is particularly desirable for clinical or environmental applications where many routine analyses must be performed each day. Reaction-rate analysis may be performed in minutes even for reactions with half- lives on the order of an hour. This same analysis would take several hours using an equilibrium based method. Reactions that are nonstoichiometric, produce unstable products or have interferring side reactions, which make them unsuitable for equilibrium-based methods, may be entirely suitable for reaction-rate analysis. Since the reaction-rate measurement can be made during the initial period of the reaction, these situations often cause no interference. Also since reaction-rate analysis is a relative measurement, constant interferences such as dirty cell windows or slightly turbid solutions do not cause errors as they would in an equilibrium-based analysis. The reaction—rate method is also advantageous in that it can be highly specific. It is possible to determine several different components in the same solution as long as their reaction rates are sufficiently different. Reaction-rate methods of analysis also have certain dis- advantages. Because only a part of the total reaction is measured, the precision of the chemical analysis is generally less than in equilibrium-based analyses. However, modern high quality measurement systems have lessened the serious- ness of this disadvantage. Another disadvantage stems from the limitation in following very fast reactions. Reaction- rate methods utilizing mixing of reagents to initiate the reaction are limited to reactions with half-lives greater than a few milliseconds. However, relaxation techniques can follow reactions with half-lives in the submicrosecond range. In addition all the parameters which affect the rate of a reaction such as temperature, ionic strength or pH muSt be controlled and/or monitored. These parameters are generally not as critical for equilibrium techniques. B. Techniques for Studying Fast Reactions Techniques for studying fast reactions must be capable of initiating a reaction and making the appropriate measure- ments in less than a few seconds. A recent study (29) includes a review of techniques for studying fast reac- tions. To be of practical value, the technique should be able to approach the millisecond time scale. Techniques which meet this criteria can be divided into two cate- gories: mixing methods and relaxation methods. The common mixing methods include continuous-flow, accelerated-flow and stopped flow. The common relaxation methods include temperature jump and pressure jump, although changes in electric field are also used. 1. Fast Mixing Methods In the fast mixing methods the reaction is initiated by the rapid mixing of the solutions containing the reacting species. The solutions flow through separate channels into a mixer where they are combined and the reaction is initiated. The reacting solution then flows into an observation cell where the reaction is monitored. The basic difference between the three fast mixing methods discussed here is in the regulation of the flow velocity and its relationship to the measurement period. In the continuous flow method, the measurement is made while the solution is flowing at a constant rate. The measurement is made while the solution velocity is being changed in the accelerated flow method. And, in the stopped-flow method, the measurement is made after the flow is stopped. The continuous flow method (13,14) developed by Hartridge and Roughton in 1923 was the first technique for studying fast reactions in solution. The reacting solutions are pushed through a mixer and then through an observation tube at a constant rate. By moving a detector along the observa— tion tube, the reaction can be observed at leisure at dif- ferent points in time. Reaction times down to one milli— second are possible with this method. However, a disadvantage is the large volumes of solution consumed, which ranges from a few milliliters to several liters. Unlike the continuous flow method, the accelerated flow method (15-19) maintains the detector at a fixed distance from the observation cell. The time profile of the reaction is obtained by varying the flow velocity. This method can be used to measure reactions on the millisecond time scale and solution consumption down to 0.1 milliliters can be attained. Its major disadvantages are a limited time scale and the added complexity of the fluid drive and flow velocity monitoring system. Because of its major importance to this work the stopped- flow method will be discussed separately in Chapter II. 2. Relaxation Methods Relaxation methods involve the perturbation of a chemical system which is at equilibrium. The perturbation causes a shift in the chemical equilibrium and the system then adjusts or relaxes to the new equilibrium concentrations. The monitoring of this relaxation is then used to determine the reaction kinetics. The perturbation is caused by an abrupt change in one of the physical parameters of the system, such as temperature (20-22), pressure (20-22) or electric field (20,22,23). These methods can be used to follow reactions with half lives on the order of nano- seconds. This greatly extends the range of reaction rates accessible for fundamental kinetics. However, there is little utility in relaxation methods for analytical pur- poses. A chemical reaction at equilibrium is utilized and initial, analytical concentrations are not involved in the relaxation expressions. CHAPTER II BACKGROUND A. The Stopped-Flow Technique The stopped-flow mixing technique was developed by Chance in 1940 (16—18). It has some basic advantages over the accelerated-flow technique which he had developed earlier. The main advantages are better reaction time ac- curacy and a wider time range available in one run. The accelerated—flow method can monitor a reaction from 1 millisecond to 10 milliseconds whereas the stopped-flow method can monitor a reaction from 1 millisecond to hours. Also, the accelerated—flow method relies on the accuracy of monitoring a varying flow rate to determine the reac- tion times at the observation cell. The stOpped-flow method only relies on the reproducibility of the time be- tween reaction initiation (mixing) and the stopping of the flow. The reaction time from that point on can be derived from any accurate electronic time base. Since the stopped-flow method has the same solution volume requirements as the accelerated-flow method, there are no realistic disadvantages of stopped-flow compared to accelerated—flow. Thus, the stopped-flow technique has essentially obsoleted the accelerated-flow method. However, there are still applications where continuous- flow methods are advantageous over stopped-flow in spite of the large solution volume requirements. This is because of the simplicity of design of the continuous-flow appara- tus and because the use of slow responding detectors to follow fast reactions is possible. 1. Principles of Stopped-Flow Mixing An ideal stOpped-flow mixer would instantaneously mix reactant solutions and immediately deliver them into the observation cell where the reaction is to be monitored. In a real system these operations require a finite amount of time. The most useful figure of merit for a stopped-flow mix- ing system is the dead time, td, which is the difference between the time of initial contact of the reactants and the time at which they are stopped in the observation cell (24-26). In a well designed system, mixing must be complete by the time the solution is stopped in the obser- vation cell. Thus the dead time must be greater than or equal to the mixing time, tm, which is the time between initial contact of the reactant solutions and "complete" mixing. A third time which is of significance because it affects td and tm is the stopping time, ts. This is the time required for flow to cease once the stopping device has begun to impede flow. For precise measure- ments tS should be much less than td. 10 a. Sequence of Operations - Figure 1 shows a pictorial diagram of a general stopped-flow mixing system with spectrophotometric detection. A controller is shown which directs the sequence of events in the system. The controller can be a manual sequencer, an electronic hard wired sequencer, a mini- computer, or a microprocessor. The sequence of operations necessary to obtain reaction- rate information by stopped-flow spectrophotometry begins with reagent preparation. Although this step is normally carried out manually, in principle all reagent preparation Operations can be carried out under the supervision of the controller. After solutions are prepared, they must be introduced into the drive system, which normally con- sists of two drive syringes. Once solutions are intro- duced into the drive syringes, the drive system is ac- tuated and the two solutions flow into a mixing chamber. The mixed solution flows through an observation cell into a stOpping device which ceases the flow after a preset flow volume or time of flow. When the flow stops, the spectrophotometric detection system is activated and data acquisition (absorbance vs. time) begins. Data pro- cessing is often carried out in order to present the data in the desired format (initial rate, concentration of analyte, rate constants, etc.). Finally the desired in- formation is presented via a readout device (recorder, print-out, plotter, etc.). 11 Figure 1. An Automated Stopped-Flow Mixing System with Spectrophotometric Detection. .«.( .L CUQQCIPU. f FEQ mNQh .H wusmfim / I u QIEZEEPL QGMFFOJQ Gimp—2.x.“ — w n I i xood us: ._zoo A mmlrm—ZOFOIQOKFUMQW S u 39.15885 tan. mun: 13 b. Influence of Dead Time on Rate Measurements - Al- though the data acquisition system begins taking data the instant the flow stops, the reaction has already proceeded for a finite time td' If td is very small compared to the reaction half-life, the reaction is essentially fol- lowed from its initiation, and the full reaction history can be recorded. In many cases (for analytical data and often in mechanistic studies), the initial reaction rate is the information of interest. This places special em- phasis on the development of systems with short dead times so that the initial rates of rapid reactions may be mea- sured. The importance of a short dead time can be deduced from a rearrangement of Eq. (3). Since the half-life, T, of a first- or pseudo-first-order reaction is given £n2 k I first order reaction and t/T << 1, by T = it can be seen that for a first-order or pseudo- I (El-(Eight = (9532—15 exp (-0.693t/T) " Egg”) (1 - 0.693t/T) (4) where, T = 0.693/k = half-life of the reaction, 3 t = reaction time, s (95%1)t = reaction rate at time t, mole 2-13-1 (d[A] _ . . . 1 . 1 “1 “'1 _HE—)o — lnltla reaction rate, mo e 2 s For the measured rate to be within one percent of the initial rate, the rate measurement would have to be 14 made within 1/69.3 seconds of the reaction initiation. If the rate cannot be measured at t << T, the analysis becomes quite complicated. The reaction time t evaluated as a function of position in the observation cell then becomes important, since in most systems the observation cell volume is a large contributor to the total dead volume. Assuming (l) plug flow of solution from the exit of the mixer to the exit of the observation cell (this is approximately true for high flow velocities in straight tubes): (2) an observation cell with uniform cross-section; and (3) the stopping time is much less than the dead time, the reaction time would be uniform for any cross-section of the cell and would be a linear function of the distance from the entrance of the observation cell. Thus, the follow- ing would hold: t 2 Itl-kEA]O exp (-kt)dt [A]o exp(-kt2 — exp(-kt1) (‘gtl)t = t = ‘t2 dt t2 ’ t1 1 (5) t -t [A]o - (d[:])t 2 1 (6) exp(—kt2 - exp(-kt1) ' (dEAJ) _ . . t t — average or measured reaction rate at time t where t - (t2 + t1)/2. t2 = the total time that the solution at the exit end of the observation cell has been mixed. 15 t1 = the total time that the solution at the entrance end of the observation cell has been mixed. For solutions with approximately the same physiCal prOperties, t1 and t2 are characteristics of the stopped- flow system and can be determined by measuring the solu- tion flow rate and the volume of the system. Then if k is known, [AJo can be determined from a measurement of d A t order or pseudo-first order. The limiting factor in this at any time as long as the reaction remains first- type of analysis is the ability of the detection system to measure the reaction rate over a small time interval. c. Character of Flow - An important aspect in the design of a fast mixing system is the character of flow. The parameter which is normally used to characterize flow is the Reynold's number, Re given by Re = gg-v where v = flow velocity, cm/s n = viscosity of the fluid, poises = density of the fluid, g/ml d = diameter of the tube, cm Flow can be classified into two categories, laminar and turbulent. The Reynold's number can be used to 16 differentiate between these two categories. Laminar flow is produced at Reynold's numbers less than 2100 whereas turbulent flow is produced at Reynold's numbers greater than 2100. This dividing line is not strictly quantita- tive but can be used as an approximate criterium in design- ing a flow system. Laminar flow is characterized by a streamline flow pattern with no eddy currents or localized transverse flow. Laminar flow in circular tubes has a parabolic velocity profile with the maximum velocity at the center and zero velocity at the walls. The pertinent equations can be derived using Newton's law of viscosity. The result is V‘, 1 _ (%)2 max thus, -l; vmax 2 where, r = the radial distance from the center of the tube R = the radius of the tube V = the flow velocity at distance r < II the maximum flow velocity max = the average flow velocity 17 In contrast to laminar flow, turbulent flow is charac- terized by the lack of a streamlined flow pattern and the presence of eddy currents and localized transverse flow. The velocity profile is also much flatter than the laminar flow velocity profile. Because of the random nature of turbulent flow it cannot be described mathematically by a straight forward application of Newton's Law of vis- cosity. Using a large collection of experimental data, the following empirical result was found to be reasonably accurate for fluids in circular tubes. 1 7 = (1 - ) .E R thus max The results on fluid flow were taken from a text on transport phenomena (27), although they can be found in any text on elementary fluid dynamics. Turbulent flow has several important advantages over laminar flow in stopped-flow and other fast mixing systems. It is highly desirable to have uniform flow velocity 1 throughout a cross-section (perpendicular to the flow) of the flow channel. This is necessary for good reaction- time resolution at the observation cell. Turbulent flow has a flatter velocity profile than laminar flow. Also 18 the transverse velocity components present in turbulent flow cause some exchange between the slower moving fluid near the wall and the faster moving fluid near the center of the tube. Turbulent flow also improves the reagent mixing process. This was demonstrated by Chance (18). He observed the seemingly paradoxical phenomenon that the point of 98 percent complete mixing moves upstream toward the mixer as the flow velocity is increased. Further advantages of turbulent flow can be seen by application of the Hagan-Poiseuille law to laminar flow and the Blasius formula to turbulent flow. The results indicate that with laminar flow in channels the flow ve- locity is proportional to the driving pressure and propor- tional to the inverse of the viscosity. However, with turbulent flow in channels, the velocity is proportional to the four-sevenths power of the driving pressure and proportional to the inverse of the one-seventh power of the viscosity. Therefore, the flow velocity would be effected less by changes in either pressure or viscosity in the case of turbulent flow. One possible disadvantage of using high flow velocities is cavitation. Cavitation is the formation of tiny vapor ' bubbles when the momentum is suddenly changed so as to cause low pressure regions in the fluid. This occurs when the direction of the fast moving fluid is abruptly changed or when upstream stopping of the flow is used. 19 Cavitation is especially troublesome with optical methods of detection, although it interferes with all methods of detection to some extent. Chance observed cavitation which originated in the mixer at high flow velocities (18). However, he was able to eliminate it up to flow velocities of 25 m/s by slightly modifying the mixer. A recent article (28) discusses cavitation in stOpped-flow systems and methods to minimize or eliminate it. 2. The Components of a Stopped-Flow Mixing System The goal of this section is to develop a perspective on the design of the components of a stopped-flow mixing system. The components include the reagent delivery and drive system, the mixing chamber, the observation cell, and the stopping device. No attempt will be made to cover every variation of each component. A recent thesis (29) has discussed the details of the components of various stopped-flow mixing systems reported in the literature. It must be kept in mind that the designs of the indi- vidual components are not independent of each other. The components must be compatible with respect to flow volume, flow rate, fluid pressure and response time. For a general purpose stopped-flow mixing system, all components which come in contact with the solution should be made from materials which are chemically inert. 20 Suitable materials of construction include glass, Kel F, Teflon and A.I.S.I. 316 stainless steel. a. Reagent Delivery and Drive_§ystem - The only type of stopped-flow drive mechanism in common use uti- lizes plungers to force the solutions from the drive syringes through the mixing system. Therefore the dis- cussion of reagent delivery and drive mechanisms will be limited to this type. Usually the reagent delivery is accomplished by drawing back the drive plungers. A double 3-way stopcock or several valves can be used to connect the sample and reagent containers to the drive syringes during this Operation and subsequently to connect the drive syringes to the mixing system. Alternatively, check valves can be used to switch connections automatically when the drive plungers are filled or discharged. The disadvantage to using check valves is that the solutions are subjected to a decrease in pressure (below atmospheric) when the drive syringes are being filled. This can cause degassing of the solutions or seepage of air around the plungers, which allows gas into the flow system. The drive mechanism must be capable of forcing repro- ducible, and usually equal, amounts of both solutions through the flow system at high pressure. In order to accomplish this, there must be a good seal between the plunger and the syringe wall, and the driving mechanism 21 must be capable of exerting considerable force. In addi- tion, the force should be reproducible and uniform through- out the drive so that accurate chemical rate measurements can be made. Suitable plungers have been constructed from stainless steel with neoprene o-rings or from Teflon with an embedded metal expansion ring. Specially con- structed gas tight Teflon syringes are available commer- cially (30). The driving force has been derived from pneumatic cylinders, hydraulic cylinders, electric motors or the experimenters hand. Pneumatic cylinders are used most frequently because they are simple, reproducible, easily adjusted and trouble free. The way in which the driving force created by a pneu- matic cylinder is reported is often ambiguous. The quan- tity often reported is the air pressure of the pneumatic cylinder, whereas the critical item is the static fluid pressure created by the action~of the pneumatic cylinder on the drive plungers. If mechanical losses are neglected the static fluid pressure can be calculated by the follow- ing equation. '1! II f static fluid pressure '11 ll air pressure in the pneumatic cylinder 5’ ll area of the plunger in the pneumatic cylinder 22 Af = sum of the areas of the plungers in the drive syringes. As an example, the static fluid pressure was calculated for two similar stopped-flow systems. At an air pres- sure of 60 psi one system had a fluid pressure of 270 psi while the other system had a fluid pressure of 750 psi. Based on previous experience, both of these systems might be reported as having a drive pressure of 60 psi! It is most important that authors take the time to cal- culate and report the static fluid pressure of their stopped-flow systems. b. Mixing Chamber - The purpose of the mixing chamber is to combine two solutions in a manner so as to produce rapid and thorough mixing with reasonable pressure drop and cavitation. Reasonable pressure drop is defined by the capabilities and limits of the rest of the flow system. Reasonable cavitation is that which subsides before the mixed solution reaches the observation cell. Rapid and thorough mixing is 99 percent mixing completed in 5 milliseconds, although somewhat less stringent mixing criteria may be acceptable. There has been no lack of creativity in the design of stopped-flow mixers. Designs have included everything from the simple "Y" mixer to the "tangential offset jets" mixer of Gibson-Milnes (31) or the "turbulent wake of a sphere" mixer of Berger (32). 23 Jet mixers are the most commonly used. They are fairly easy to construct and can offer good mixing efficiency. There is a variety of designs including opposed jets, tan- gential jets and a compromise between the two. Chance found the compromise produced the least turbulence (18). Berger (33) found that the optimum offset between the pairs of tangential jets was one jet diameter. A general rule of thumb to avoid cavitation and excess pressure drop is the total area of the jets into one chamber should be equal to the area of the main flow channel. Increas- ing the number of jets will increase mixing efficiency if the flow rate is maintained. However the pressure drop will increase. In designing a mixer, there is always a trade off be- tween mixing efficiency on the one hand and increased pressure drop and cavitation on the other hand. c. Observation Cell - The construction of the observa- tion cell depends on the type of detection used. The only general criteria are that the volume should be small and that the cell should be constructed so that cavitation is avoided. Also, a cylindrical observation cell is best from a fluid dynamics point of view. In spectrophotometric measurements, the path length will be important. It is desirable to have a long path length for good sensitivity, but a short path length gives better time resolution and a shorter dead time. 24 In molecular fluorescence, an observation cell with a square cross-section may be preferred. And, if conducto- metric measurements are to be made, the cell must be con- structed of nonconducting materials except for the elec- trodes. d. StOpping Device - The stopping device must be able to stOp the flow rapidly without causing shock waves to propagate through the flow system. The time from when the device first begins to impede flow until it stops the flow completely should be much less than the dead time. It is difficult to find valves which close in a fraction of a millisecond. However, one was develOped by Berger and coworkers (34). The most common type of mechanism utilizes a stopping syringe. The plunger of the syringe comes up against a fixed block thus causing an abrupt stop. The block and/or the plunger tip should be made of a relatively soft metal to prevent the formation of shock waves in the solution. Sturdevant (35) caused stopping upstream by the contact of tapered pins rather than a blunt stOp block. e. Detection and Readout System - Virtually any method- of detection which has a response time on the millisecond time scale and can be used on small sample volumes can be applied to a stopped—flow mixing system. UV-visible spectrophotometry is the most common mode of detection. 25 The major improvements with this type of detection have been in the development of high intensity stabilized light sources (36-38). Several workers have utilized monitoring of the light source intensity in order to obtain highly accurate spectrophotometric data (37,39). Scanning spectrophotometric systems have been developed in order to follow transient species. These systems utilize a rapid scanning monochromator (40-43) vidicon tubes, or diode arrays (44). In order to be useful, the system must be capable of scanning a spectra in a few milliseconds. Photomultiplier tubes (PMT) or photodiodes are the two types of transducers used with spectrophotometric detection. The transducer which gives the best noise- drift characteristics has been the subject of consider- able debate (45-50). The photodiode-high gain amplifier combination has superior characteristics at high light levels, but at lower levels the PMT-low gain amplifier combination gives better results. Other means of detection include molecular fluores- cence (51), light scattering (52-53), IR absorption (54), ESR (55) thermal methods (34) and electrochemical methods (56-58). Readout devices for stopped-flow systems range from a storage oscilloscope equipped with a Polaroid camera to a minicomputer system including mass storage, CRT display and teletype. Numerous analog and digital hardware rate 26 meters have been developed for specific applications. The various types of readout systems have been recently reviewed (59,60). 3. Manual Stopped-Flow Systems The development of stopped-flow systems up to 1972 has been covered in a recent thesis (29) and will not be re- peated here. Also systems in which both the operation of the mixing system and the data acquisition and analysis have been automated are discussed in the next section. A stopped-flow system with accurately controlled thermo- stating and a flow system entirely of glass or quartz has been described by Caldin and coworkers (61). The entire flow system except for the drive unit is emersed in a thermostating bath. Temperatures from -10 to +55°C have been used. It requires approximately 10 minutes for tem— perature equilibration. Flexible fiber optics transfer light to and from the observation cell, which has a 2 mm path length. The authors report a 3-4 ms deadtime (determined by the extrapo- lation method) even though a simple 2-jet mixer is used. A storage oscilloscope is employed as the readout device.- Peterson and Mock (62) have described the use of a commercially available dual wavelength/split beam stopped- flow spectrophotometer for analysis of turbid samples. In the dual wavelength mode, accurate determinations 27 can be made on reactions with half-lives down to 50 ms. A PDP ll/OS minicomputer is used for data acquisition and analysis. A variable temperature, rapid scanning stopped-flow spectrophotometer was reported by Dye and coworkers (40-42). The system is emersed in a thermostating bath for thorough temperature control. The entire flow and reagent delivery system is constructed of glass and Teflon and is vacuum tight for work with air sensitive solutions. The flow system requires a minimum of 2 ml of each reagent and 0.75 ml are used per run. The detection system is double beam to cancel out source and PMT fluctuations. A high intensity, 1000 watt xenon arc lamp is used to obtain high light levels and high signal-to-noise ratio (S/N). Quartz flexible fiber Optics lead to and from the reference and Observation cells. The system contains two Observation cells (0.199 cm and 1.85 cm) to allow measurements of a wide concentra- tion range. Dead times of 2.7 and 6.7 ms were determined for the short path length cell and long path length cell, respectively. The scanning system consists of a modified Perkin- Elmer model 108 rapid scan monochromator. The modifica- tions involve optoelectronic transducers to encode the position and velocity of the scanning mechanism. A phase- locked loop frequency multiplication system allows the data sampling rate to be synchronized with the scanning 28 monochromator. The system can scan up to 150 spectra per second. The detection system is interfaced to a PDP 8/I mini- computer for data acquisition and analysis. The inter- face is capable of sending parallel digital data over several hundred feet at rates up to 10 MHz. The data points are averaged to provide a bandwidth which can be varied with time to optimize the S/N. Averag- ing is also desirable because it economizes the use of computer memory. The display of time dependent spectra via a high speed CRT display provides the experimentor with on-line feedback. A second computer interfaced rapid scanning stopped- flow spectrophotometer was reported by Wightman and co— workers (43). The computer is used to control the scan and acquire and analyze data. The system employs a com— mercially available rapid scanning spectrometer (RSS) which was developed by Kuwana (63). The RSS accomplishes rapid scanning by the rotation of a mirror attached to a galvanometer armature. The instrument was modified so that the voltage to the galvanometer was supplied by a computer-controlled digital-to-analog converter. The system is capable of taking a 50 point, 250 nm spectrum in 1.2 ms with a repetition rate of 2 ms. The stopped-flow mixing system is also a modified com- mercial unit. This system has an electric motor drive system. The flow system was modified from downstream 29 stopping to upstream stopping. To prevent cavitation caused by upstream stopping a pocket of air at the exit end is compressed during flow to create back pressure. About 1 ml of each reactant solution is required per run. 4. Automated Stopped-Flowygystems Automation of rate measuring systems is an area of vigorous research (59,60). As will be demonstrated in this section, stopped-flow systems have certainly received their share of the attention. The Operation Of a complete stopped-flow system can be divided into three parts: (1) sample and reagent preparation: (2) mixing and flow stoppage and (3) data collection and analysis. Automation of the entire system not only reduces human labor tremendously, but for fast reactions it reduces the analysis time by several orders of magnitude. The major time efficiencies occur in the automation of steps (1) and (3), but auto- mation significantly reduces human labor in all three steps. Another inherent advantage of automation is better reproducibility. Automation can be accomplished by analog-digital hardware, but the use of a small computer to control the entire system greatly improves efficiency and flexibility. This discussion on automated stOpped-flow systems will be restricted to those systems in which the automation of both the mixing system and the data analysis have 30 been accomplished. One of the first systems to have automated sample hand- ling as well as automated data handling and operation of the instrument was developed by Javier and coworkers (64). The automatic sampling system consists of a motor driven sample turntable and a sample introduction system based on a rapid injection and automatic refill pipet (65). The rapid injection pipet also serves as the drive mechanism for the stopped-flow mixing system. One cycle of the sampling system takes less than one second. The sampling system can be operated manually or programmed to Operate in synchronization with the stopped-flow system. The spectrophotometric observation and readout system utilizes a stable high intensity tungsten lamp as a light source and accomplishes automatic readout with a digital ratemeter. The average of ten results can be read out on the digital display in 10 seconds, which includes cell flushings between samples. Another automated stopped-flow system utilizing a hardware rate meter was reported by Beckwith and Crouch (66). The basic flow system consists of two delivery syringes with a pneumatic drive, pneumatically operated delivery and waste release valves, a mixing chamber of the Gibson-Milnes design, an Observation cell for spectro- photometric detection and a spring-loaded stopping assembly. Vertical flow was used to eliminate air bubble problems. 31 The entire operating cycle is controlled by a digital sequencing system. Initial rates are measured automatically using a fixed- time digital readout system (67). The rate meter can measure both positive and negative slopes and has a dynamic range of over four orders of magnitude. Approximately 1000 samples can be analyzed per hour, including changing solutions manually and several rinsing steps between samples of different concentrations. The first reported use of a small digital computer for stopped-flow-data acquisition and evaluation was by DeSa and Gibson (68). They utilized a DEC PDP 8/I com- puter with a fast-analog-to-digital converter plus external control and timing circuits. A standard Durrum stOpped- flow spectrophotometer was used except the hydraulic drive was replaced with a pneumatic cylinder. The data acquisition involves taking 400 samples of the response curve at accurately known time intervals. The sampling rate can be varied from 1 Hz to 2 x 104 Hz and there is an option of changing the sampling rate during data acquisition. This allows some optimization of sampling rate with a changing response. After data acquisition, the computer checks for over- flow of the range of the analog-to-digital converter. If overflow did not occur, smoothing of the data to reduce noise effects can be carried out. Mean values and stan- dard deviations are calculated from replicate runs and 32 the results outputed as printed copy and/or punched paper tape. In cases where smoothing is not required, the computer can be instructed to take only 20 data points. This results in the capability Of representing a response curve with points as close as 50 microseconds apart. This system is capable of making absorption and f1uores~ Cence measurements and has been applied to problems in- volving multiple inputs as in polarization of fluorescence and dual wavelength measurements. An automated computer-controlled stopped-flow system utilizing a novel mass-based solution preparation system was recently reported by O'Keefe and Malmstadt (39). The stopped-flow mixing unit fits into a modular spectrophto- metric setup (69). A single TTL pulse from the computer causes the stopped-flow mixer to perform one complete cycle. Desirable features of the mixing system include small sample volumes (0.17 ml) and temperature monitoring of the reacting solution via a high speed thermistor. Highly accurate spectrophotometric measurements are made with a high intensity unregulated light source. This is accomplished by monitoring the light intensity via a beam splitter and second photomultiplier tube. The solution preparation system (70) is based on the weights of solutions rather than their volumes. An elec— tronic weight sensor monitors the weight of the sample vials on a turntable as each solution is prepared. The 33 system is simple and accurate, but the dynamic range of the concentrations prepared is limited. The computer controls the operation of the entire system from solution preparation to data analysis. The experimentor has a choice between a routine mode of Opera- tion for reaction-rate analysis and an investigative mode of operation for fundamental studies. A highly automated stopped-flow system was developed by Sanderson and coworkers (71). The system consists of a sample preparation unit, a sampling unit, a Sturdevant- type stopped—flow mixer, an Optically stabilized spectro- photometer, and a small computer (Hewlett-Packard 2115A). The computer is used to control the instrument and to analyze the reaction-rate data. Instructions are given to the computer as to what samples are to be prepared and in what order they are to be prepared, the rate at which data is to be taken, the amount of data to be taken, a delay time, a noise incre- ment and what mathematical Operations are to be performed on the data. The system delivers desired amounts of sample solu- tions and diluent to a receiving vial. This mixture is then transferred to the stopped-flow to be rapidly mixed- with reagents and delivered to the observation cell. Spectrophotometric data are collected and processed by the computer. The results are displayed on an oscillo- scope or printed out via a teletype. 34 The computer software is divided into two programs. First a "set-up program" is used to control the sample preparation and then an "operational program" is employed to perform data acquisition and analysis. In order to initiate sample preparation, stock concentrations of the individual constituents along with the volume of the receiving vial are entered as parameters via the teletype. The computer then asks for the concentrations to be used for each run. The data acquisition and analysis procedure was des- cribed by Willis and coworkers (38). The analog-to- digital converter (ADC) can take data at rates from 0.1 Hz to 104 Hz. The resolution of the lO-bit ADC was im- proved tO between 14 and 15 bits by utilizing a real-time variable offset method described by Deming and Pardue (72). The data processing method provides for operator inter- action if desired. The operator has the option of dis- playing the unprocessed transmittance data on an oscillo- scope for diagnostic purposes. Any given run can be re- jected and the Operating conditions changed if so desired. The program calculates absorbance information and can determine an apparent first-order rate constant at each of several successive points based on a 21 point least squares slope. Display of these slopes provides a check on the first-order assumption. At a later stage the program can determine a more accurate rate constant computed as the least squares slope over one half-life of the reaction. 35 Analysis of an unknown is performed by determining the ratio of the rate of change of absorbance of the un- known to that of a standard. This assumes first-order behavior. The most sophisticated stopped—flow system reported to date (73) was developed in the same laboratory as the previous system (71). It utilizes the same stopped-flow spectrophotometer as its predecessor, but the solution preparation unit and the computer system are different. The control system is a heirarchical arrangement in which a minicomputer directs a microcomputer to prepare reagents and operate the stopped-flow mixer. Thus, the minicomputer is freed of these time consuming tasks which do not utilize its computational power. In addition to sending directives to the microcomputer, the minicomputer acquires and analyzes data and designs new experiments based on simple criteria. The data acquisition routine uses an exponentially changing Clock rate for optimum signal-to-noise ratio. The sample preparation unit uses a parastaltic pump to deliver the prescribed amounts of solutions to a sample turntable. The authors claim greater versatility for the new sample preparation unit, although the previous unit, which utilized micrometer driven syringes, had somewhat better accuracy. 36 B. The Chemistry Of 12-Molybdophosphate The lZ-molybdophosphate anion (lZ-MPA) is formed by the reaction of phosphate with cationic molybdenum(VI) species in strongly acid solutions. The study of the for- mation Of lZ-MPA is complicated by the lack of knowledge of the exact form of the molybdenyl species. Several recent studies (29,74) have reviewed the pertinent work on the chemistry of lZ-molybdophosphate and molybdenum(VI). A brief summary Of this work including any recent develop- ments will be given here. 1. Molybdenum(VI) in Aqueous Solution Molybdate dissolves in basic solution to form the Moo:- species. However, as the pH is decreased toward the iso- electric point, a variety of Mo(VI) species are formed. The techniques of spectrophotometric and pH titrations (75,76), Raman Spectroscopy and ultracentrifugation (76), enthalpy titrations (77), and dialysis and electromigration (78) have been used to identify these species. Sasaki and Sillen have reviewed the work in this area (75). At the isoelectric point insoluble molybdenum trioxide (M003) forms. Upon further acidification, this precipitate re- dissolves as a cationic species. The exact form of the anionic or cationic molybdenum species depends on the bound acid-to-molybdate ratio (Z). However, investiga- tions by numerous workers have resulted in conflicting 37 results. Studies (79-82) have indicated that the first step in 4 tion which involves a change in coordination to octahedral the protonation of tetrahedral MOO is a two proton addi- 0MO(0H); or Mo(OH);. However, recent evidence suggests the protonated form exists as the less symmetric cis-dioxo M002(H20) (OH);. At a bound acid-to-molybdate ratio (2) of about 1.14 there is a rapid condensation of this monomer to the well known heptamer, M0703;- At about Z=l.5 various workers (76,81,84) have found 4- evidence for the formation of an octameric species, M08026, which can be isolated in the solid state. However, the results of other workers (75,83,85) have not supported this contention. As Z is increased above 1.5 further protonation occurs and eventually MOO3 precipitates at the isoelectric point. If still more acid is added, MOO dissolves as a cationic 3 species (86). Both monomeric (78,87) and dimeric (75,88, 89) molybdenyl species have been suggested. The work of Krummenacker and coworkers (90-94) indi- cates the presence of protonated and unprotonated monomeric and dimeric cationic species. Formation constants were determined as a function of solution acidity (93,94). Other workers (95) found evidence for a singly charged monomeric species and doubly charged dimeric species at perchloric acid concentrations greater than 3M. A recent study (87) determined equilibrium constants for the 38 protonation and dimerization of molybdenyl species in agreement with Krumenacker. 2. The Formation of 12-Molybdophosphate Twelve-molydephosphate is the most widely studied heteropolymolybdate compound. Both the solution chemistry and the solid crystal structure have been studied. The molecular formula for the solid has been determined (96,97) as H3M012PO40 with 29-31 water molecules, six of which are structural. The MOO6 octahedra surrounding the P04 tetra- hedron have been shown to be somewhat distorted (97). The formation of lZ-MPA has been studied by numerous workers. Crouch and coworkers (89), who studied the re- action in nitric and perchloric acid solutions, found evidence in agreement with the overall stoichiometry sug- gested by Souchay (88), viz., H po + 6HM o+ + 12-MPA + 9H+ 34 °26+ However, they found a slightly different stoichiometry in sulfuric acid. Other workers have postulated a different stoichiometry starting with a molybdate instead of a molybdenyl species (98). Halasz and Pungor studied the formation and decomposi- tion of 12—MPA (99). These authors postulated the pH dependent formation of two different forms of lZ-MPA 39 similar to those found (100) for lZ-molybdosilicate (12- MSA). However, no spontaneous transformation between the two forms was found as in the 12-MPA case. Crouch and coworkers (89) concluded that the formation of lZ-MPA involves an initial reaction of phosphate with the molybdenyl cation followed by several condensation steps. The reaction was always first order in phosphate, whereas it varied from first to sixth order in Mo(VI) depending on the acid-to-molybdate ratio. The rate varied from zero order in acid at low acid concentrations to inverse eighth order at high acid concentrations. The formation of lZ-MPA has been studied using proton and 31P NMR (101,102). Also, numerous studies have been undertaken concerning the reduction of 12-MPA to the hetero- poly blues (89,103-107). CHAPTER III THE COMPUTER-CONTROLLED STOPPED-FLOW SPECTROPHOTOMETER In this work a first-generation stopped-flow spectro- photometer (66) has been completely revamped and inter- faced tO a PDP 8/e minicomputer. The modifications include: installing quartz optics between the monochromator and the observation cell and between the observation cell and the photomultiplier tube (PMT); (2) thermostating the entire system including the drive syringes and the reagent con- tainers: (3) inserting a manual valve between the mixer and the observation cell: (4) designing a more efficient, easily replaceable mixer; (5) inserting a fast thermistor probe in the flow stream just below the observation cell; and (6) installing an Optoelectronic trigger module. An extensive set of computer programs has been developed to Operate the instrument and to acquire, analyze and dis- play the spectrOphotometric and temperature data. A block diagram of the instrumental system is shown in Figure l. The computer controls the Operation of the stopped-flow mixing system via three TTL-switched optically isolated power relays (108). At the end of a push, a trigger signal is sent to the computer to initiate data acquisition. This signal is the sole synchronization link between the instrument and the computer. 40 41 The radiant power coming from the spectrophotometer (light source and monochromator not shown as separate units) is converted to a current via a 1P28 photomulti- plier tube. The current is then converted to a voltage by a variable gain current-to-voltage (I/V) converter. Finally the voltage is converted to a binary number via the 12-bit analog-to-digital converter (ADC). The computer accepts this digital input and subsequently performs data analysis and outputs the results via a printer, a plotter or a fast CRT terminal. The data are also stored on a magnetic disk (not shown) for future use. The fast CRT display is particularly valuable in that it provides on— line feedback. A. The Stopped-Flow Mixing System The stopped-flow mixing system consists of a reagent delivery and drive mechanism, a mixer, an observation cell and a stOp syringe. Each of these parts will be discussed and then the operation of the stopped-flow mixing system as a whole will be explained. The system is shown in Figure 2. A description of the designated parts follows. A, B, M:‘ Air cylinders. These control, respectively, the drive plungers, valve D and valve J. C: Drive syringes D: Double 3-way stopcock valve E: Reagent inlet tubes 42 Figure 2. A Stopped-Flow Spectrophotometer. 43 "BE-.524 mag—.40): 0.? uhzwmmnu wank 191.2533! nOFOIm I COF<20¢IU -0202 wumam PIG... Figure 2. 44 F: Mixer block. This contains the mixer and a manual valve after the mixer to prevent diffusion of unmixed reagents into the observation cell. G: Flexible quartz fiber optic light guide. H: Observation cell. I: Quartz rod coated for internal reflection. J: Waste release valve, single 3-way stopcock. K: Solution exit tube. L: Stop syringe. M: See A. N: Photo-interruptor module for trigger signal. 1. Reagent Delivery and Drive Mechanism The purpose of the reagent delivery and drive mechanism is to deliver the reagents into the drive syringes and then to force them from the syringes through the flow system. The flow of reagents into and out of the drive syringes is caused by movement of the plungers via the pneumatic cylinder. 'The plungers are stainless steel with double neoprene O-rings. The direction of flow is controlled by the double 3-way stopcock. In one position Of the stopcock the drive syringes are connected to the reagent tubes, whereas in the other position the syringes are connected to the flow system. When filled, the syringes contain enough solution for approximately six pushes (0.45 ml per push) with good purging of the solu- tion from the previous push. The amount of solution used 45 per push is controlled by the stop syringe, which will be discussed later. The important factor in determining the velocity of the fluid in the flow system is the static fluid pressure and not the air pressure in the pneumatic cylinder. However, as indicated in Chapter two, any back pressure at the exit end of the flow system must be subtracted from the static fluid pressure in order to obtain the true fluid driving pressure. This will be discussed further under "stopping syringe and trigger". The static fluid pressure is created by the force of the drive syringe plungers on the solu- tions. Since the 3/8" drive plungers are rigidly attached to the 1 1/8" pneumatic cylinder, the static fluid pres- sure is equal to 4.5 times the air pressure in the pneu- matic cylinder. At a normal air pressure of 75 psi, the static fluid pressure is 338 psi. 2. Mixer The first-generation stopped-flow originally had a tangential jet double mixer commonly used for stopped- flow applications (31). However, the mixing efficiency appeared to be poor. It was observed that no decrease in the dead time of 7.5 milliseconds was realized as the air pressure was increased from 60 psi to 90 psi. This pressure increase should result in an increase in flow velocity of more than 25 percent and a concomitant decrease 46 in the dead time. The dead time was determined by the extrapolation technique using the iron-thiocyanate reaction (24). At this point the alignment and other mechanical aspects were checked to make sure this Observation was not due to some mechanical problems. Having determined that there were no mechanical problems, it was concluded that the mixer should be replaced. Two simple designs were tried, but they gave essentially the same results as the original mixer. The third mixer which was tried is shown in Figure 3. This mixer gave a dead time of 5.5 milli- seconds at an air pressure of 70 psi and a dead time of just under 4 milliseconds at 90 psi. The mixer was designed for easy replacement in the event that it proved unsatisfactory or for cleaning pur- poses. The mixer simply slips, very snuggly, into a cylindrical opening. Except for the entrance and exit, the flow is between the mixer body and the walls of the opening. The two solutions come together at the top of the mixer and then flow out of the entrance channel through the four upper holes. Mixing then continues around the perimeter as the solution is forced to flow around the lugs which seal against the containing walls. Finally, the solution flows into the four lower holes and out the exit channel toward the Observation cell. 47 MIXER VOLUME=OC446cm3 Figure 3. The Stopped—Flow Mixer. as we struc leads throu at a Kel F The c. elect: are t} Press The 0; insert Pathle A t inst k miStoz The p1 bead j The Stop t: the pl until 48 3. Observation Cell The Observation cell is designed to enable conductance as well as absorbance measurements. The cell was con- structed by sandwiching four-3 mm platinum disks (with leads) between blocks of Kelf‘and then boring a 2 mm hole through the stock. Entrance and exit channels were bored at a right angle to the cell channel at either end. The Kel F sandwich is fitted into a stainless steel housing. The cell is shown in the upper left of Figure 4. The electrodes are not shown. Stainless steel inserts, which are threaded on the inside and outside, are used to com- press the Kel F sections in order to seal the electrodes. The Optics, also shown in Figure 4, are screwed into the inserts and seal the ends of the observation cell. The pathlength of the cell is approximately 2 cm. A threaded thermistor port intersects the flow channel just below the Observation cell. A fast responding ther- mistor is epoxied into a stainless steel threaded plug. The plug is screwed into the port so that the thermister bead just protrudes into the flow channel. 4. Stopping Syringe and Trigger The purpose of the stopping syringe is to suddenly stop the flow of solution. The flow of solution causes the plunger of the stopping syringe to be pushed back until it comes in contact with the stop block. At that 49 .mofiuao mpnmso ocm HHOO COwuo>uwmnc BonIowamoum one .q wusmfim ooze“. 053°... .m.m 98 22:5 820 toen— ntoao 29.6. .1 36525052 :ou cozoiomno 059.6: 5:8 98 com 2.6.30 “.203 @ //fi11<1111// Ifi~httsix |-,-/// 50 point the flow is suddenly stopped. Judging from the amount of "rounding" of the absorbance trace of a fast reaction, the time required to completely stop the flow is less than a tenth of a millisecond. The stop block consists of a threaded bolt so that the amount of flow before stopping is adjustable. The plunger to the stopping syringe is spring loaded so that when the waste release valve is switched from the flow to the waste release position, the spent reagents are ejected automatically. An important consideration is the amount of back pressure caused by the loaded spring. The spring force was measured by suspending a weight from a rod which was attached to the syringe plunger on one end and rested on a pivot on the other end. The spring force is 5.7 pounds at the beginning of the push and 9.1 pounds at the end of the push. This translates to back pressures on the fluid Of 74 and 119 psi. In the section concerning the drive mechanism it has been shown that the normal static fluid pressure is 338 psi. Thus the fluid driving pressure is 264 psi at the beginning of a push and 219 psi at the end. In this work the stopping mechanism was modified slightly to include a triggering mechanism. The trigger signal must occur at a reproducible time prior to the end of a push so that the data collection can be synchronized to the time that the flow stops. The triggering mechanism was constructed as follows. 51 A hole was bored through the center of the stop block (bolt). A rod was inserted through this hole and attached to the stopping syringe plunger. A photo-interruptor module (G. E., Model H13Bl) was mounted so that the end of the rod interrupts the light path just before the plunger comes to rest against the stOp block. The time between the trigger signal and the actual stopping time (trigger time) can be varied by changing the position of the photo-interruptor module. This may be useful in characterizing the stopped-flow mixing system. However, under normal circumstances, the position would remain fixed. Of course the trigger time also depends on the driving pressure. Using an air pressure of 75 psig, the trigger time was 10.0 milliseconds. The trigger time was determined as the time (from the trigger signal) at which the absorbance trace of the iron-thiocyanate reaction begins to deviate from its level value during flow. This determination was performed several times on different days with a reproducibility of better than 0.2 millisecond. 5. Sequence of Operations A description of the Operation of the mixing system as a whole follows. See Figure 2 for locations of the components being referenced. (1) Valve "D" is switched to allow the reagents to be drawn into the drive syringes. 52 (2) The drive syringe plungers "C" are drawn back via pneumatic cylinder "A". Then valve "D" is switched back to connect the drive syringes with the flow system. (3) The pneumatic cylinder "A" is then pressurized in the downward direction to force the solutions through the flow system. However, no flow occurs because valve "J" is in the waste release position. (4) Valve "J" is rapidly switched to the flow posi- tion at which point solution flows through the entire system pushing back the stop syringe plunger "L". (5) Just before the stop syringe plunger hits the stop block, the rod which is attached to the plunger passes between the LED and the phototransistor of the photo-interrupter module. This causes the trigger signal to synchronize the data taking to the time at which the stop occurs. The flow stops shortly there- after, typically 10 milliseconds. (6) After the data are taken, valve "J" is switched to the waste release position whereupon the spent solution is expelled through tube "K". (7) Operations (4) through (6) are repeated until the drive syringes are empty. normally six times. B. The Spectrophotometric Detection System The components of the spectrophotometric detection system are illustrated in Figures 1 and 2. These 53 components are listed below. (1) GCA/McPherson, EU-701-50 Light Source. (2) GCA/McPherson, EU-700 Monochromator. (3) Schott Optical, 25 cm, 2 mm diameter, quartz fiber optic bundle. (4) Custom built Observation Cell (109). (5) Schott Optical, QLG, 10 cm, 3 mm diameter quartz rod, coated for internal reflection. (6) RCA, 1P28 Photomultiplier Tube and Heath, EU-42A High Voltage Power Supply. (7) Keithley, 427 Current Amplifier. (8) Heath, EU-800-GC, D/A Converter Card; utilizing an Analog Devices, MDA 102-25, 10-bit D/A converter. (9) Datel, DAS-16-M12B, lZ-bit, 8—channel A/D Converter. The specifications for these components and other equip- ment used in this work are given in Appendix A. The non- standard items in this list are the Observation cell, the quartz optics and the Offset circuit. The observa- tion cell has been discussed previously, and the other two items will be discussed here. The optics were designed with two purposes in mind, viz., to maximize light throughput and to eliminate the disturbances caused by any vibrations of the stopped- flow mixing system. These vibrations are caused by the rapid switching of the waste release valve and by the stop syringe plunger striking the stOp block. Since the 54 stopped-flow spectrophotometer was built as a modular system, these vibrations cause disturbances in the Optical transmission between the monochromator and the observation cell. Since the PMT is bolted directly to the mixing unit, vibrations present no problem there. The Optics are shown in Figure 4. The 2 mm diameter flexible quartz fiber optic bundle which couples the mono- chromator to the observation cell has several advantages. Since it is rigidly fixed to both units and since its transmission is not affected by slight bending, the vibra- tion problem is eliminated. The light throughput is quite good for several reasons. The fiber bundle diameter is the same as both the observation cell diameter and the usual slit width Of the monochromator. The internally reflecting aspect of the fibers greatly reduces light losses. Light losses are further reduced since the end of the fiber bundle forms the window of the observation cell. This eliminates a quartz-air interface which would reduce transmission by about 10 percent due to Fresnel reflections. Since vibrations are not a problem between the observa- tion cell and the PMT, a 3 mm diameter, internally reflect- ing, quartz rod was utilized there. The rod must be slightly larger than the diameter of the Observation cell because it forms the end seal. However, this causes no light loss since it is the (light) exit end of the observation cell. In the case of the fiber bundle, the 55 stainless steel sheath around the bundle forms the seal. Divergence losses are reduced in that the rod transmits the "trapped" light right up to the window of the PMT. The rod has the added advantage that the cross-sectional area is 100% transmitting as Opposed to about 70% for the fiber bundle. There was approximately a one hundred-fold increase in light intensity by adding these Optics to the system. This was determined by the increase in the photocurrent at the same PMT voltage. The other improvement in the detection system involves automatic, calibrated offset of the photocurrent to enable scale expansion measurements. The 12-bit ADC limits the resolution Of signals to the computer to 1 part in 4000. However, with accurate Offset and scale expansion, spectro- photometric resolution of 1 part in 200,000 with better than 1% accuracy has been achieved. The Keithley 427 Current Amplifier was modified so that offset can be performed remotely. The normal method of Offset is via a potentiometer, which is connected between the +15V and -15V sources within the current amplifier. The center tap of the potentiometer is connected to a resistor which leads to the summing point Of the opera- tional amplifier. Decade values for the summing point resistor are selectable via a switch on the front panel of the current amplifier. A single-pole-double-throw switch was inserted in the circuit so that the summing 56 point resistor can be connected to the potentiometer for manual offset or to a BNC connector for remote offset. The remote offset voltage is supplied by a Heath EU-800- GC, 10-bit digital-to-analog converter (DAC) followed by an Analog Devices AD518K Operational amplifier used as a unity gain inverter. Since only negative currents are produced by the PMT, only positive offset voltages are required. The DAC is controlled by the computer. C. The Thermistor and Thermostating System The entire flow system is thermostated by circulating water from a Neslab T.V. 45/250 thermostating bath. The temperature of the water in the 50 liter bath is con- trolled to 0.l°C. The temperatures from 10°C to 40°C have been used with no difficulties. The solution contain- ers and the drive syringes have thermostating jackets. The rest of the flow system has numerous channels bored through the stainless steel blocks for thermostating purposes. However, thermostating cannot control solution tempera- ture changes on the millisecond time scale. Fast solu- tion temperature changes can be produced by heats of re-_ action or dilution and by viscous heating of the flowing solutions. The temperature rise due to viscous heating of water flowing in the stopped-flow mixing system was measured by a fast thermistor and found to be a few tenths of a degree centigrade. 57 The rate of temperature equilibration in the 2 mm di- ameter Observation cell can be calculated as follows. For a tube of circular cross-section and constant wall temperature (perfect thermostating), the heat flux through the wall is given by, Q = hAAT, cal-sec"l where h = heat transfer coefficient, cal-cm"2°s-]"°C-l A = total wall area, cm2 AT = temperature difference between the fluid and the wall, °C. The value of h for free convection in water (still water) can be Obtained from "Perry's Chemical Engineer's Handbook, 4th Edition, 10-20. It is 0.004 for a tempera- ture difference of 0.1 to 0.5°C and goes up to 0.008 for a temperature difference of 5°C. The normal tempera- ture difference would be less than 0.5°C, so the value of 0.004 for the heat transfer coefficient is used here. The rate of temperature equilibration, RTE, can be cal- culated as follows: RTE = MTT' . I S 58 where, m the mass of the fluid, 9 l 1 ~°C" . c the heat capacity of the fluid, cal-g- The values for water in a 2 mm diameter by 2 cm long cell are: h = 0.004 cal-cm-2:sml-°C-1 A = 1.26 on2 m = 0.063 g c = 1.0 cal-g"1-°C-l Thus, 1 RTE = 0.08 s- or 8% per second. Therefore the temperature of the solution would be approaching the wall temperature at a rate equal to 8% of the difference per second. Assuming that the tempera- ture of the solution remains uniform through free convec- tion, it would approach the wall temperature in a decay- ing exponential fashion. The rate is Obviously too slow to assume temperature equilibration for fast reactions. Because of the inability to control temperature pre- cisely, a fast responding thermistor (7 ms time constant) was installed as described earlier. The thermistor and associated monitoring electronics were recently discussed (110). D. Computer Interface All digital signals to and from the PDP 8/e computer are transferred through a Heath Computer Interface Buffer. 59 A Heath EU-801-21 I/O Patch Card is used to transfer data and timing pulses between the peripherals and the com- puter. The digital logic used to Operate the stopped- flow system via signals from the computer consists of an octal decoder, NOR gates, NAND gates and flip-flops. The flip-flops hold the logic states for the Optically iso- lated power relays which control the operation of the stopped-flow mixing system as explained earlier. The DAC, used for offset, is operated by signals from the I/O Patch Card. The logic and interface design for the ADC has been explained in a recent thesis (111). All the inter face cards except thosed for the ADC are housed in and powered by a Heath EU-BOlI Computer Interface ADD. E. Software The software to operate the stopped-flow mixing system and acquire and analyze the spectrophotometric and tempera- ture data was designed for maximum flexibility. The DEC OS/8 operating system allows chaining between programs with data and other parameters saved in a "common" area of the computer memory. Chaining involves the use of a file oriented mass storage device. The computer system I used here has a DEC RK05 disk drive and also a SYKES dual Floppy Disk drive for mass storage. The program which is chained to is swapped into memory and its execution begun automatically. In this manner a closed loop 60 computational system involving many separate programs can be created. In this work assembler language (PAL8) programs were created to operate the stopped-flow and acquire raw data related to absorbance and/or temperature. After the data are acquired, a FORTRAN program is swapped into memory. The FORTRAN program converts the raw data to absorbance and/or temperature, stores the data on the disk, analyzes the data and displays it if desired. The experimenter can then have the computer chain back to the PAL8 program to acquire more data or chain to some other program or exit. For maximum control all chaining is specified in real time by the experimenter. FORTRAN programs are also avail- able for Off-line analysis, display, etc. of the data files stored on the disk. Appendix B lists the main programs used in this work along with a brief description of their capabilities. Four different PAL8 programs were written to cover the Options of taking temperature data along with the spectrophotometric data and using scale expansion for the spectrophotometric detection. The PAL8 program which includes scale expansion option is given in Appendix D and the dialog is given in Appendix C. The FORTRAN program which this PAL8 program would chain to is shown in Ap- pendix E. The calculation of absorbance values when using the scale expansion mode is somewhat complicated and will 61 be explained here. The offset is adjusted and calibrated and then the raw data (ADC readings) are acquired by the PAL8 program. The raw data and offset parameters are then passed to the FORTRAN program where absorbance values are calculated. The Offset calibration and data collection are per- formed as follows: (1) The operator selects the amplification and offset range on the Keithley: (2) With the light source shutter Closed, the dark current level (ZRLVL) is recorded. Then the computer varies the offset and records the output levels in order to obtain the factor relating the change in the DAC.setting. to the change in the ADC reading. This factor is stored as a binary number (FCTR) and a binary exponent (FEXP). (3) The shutter is Opened, and the Offset is adjusted so that the blank level is just below the upper limit of the ADC (+5V). This ADC reading (BLNK) is saved along with the DAC setting (UDAS). (4) The sample is run, and the ADC readings (PV) stored as raw data. (5) The FORTRAN program is swapped into core with the Offset parameters, and the blank value saved in "common" along with the raw data. The transmittance range (TR) can be calculated in terms of the ADC readings as follows: 62 FEXP TR = (UDAS)(FCTR)(2) + BLNK - ZRLVL The transmittance of a data point (TP) can then be written as, (BLNK-PV) TR H H PT For computational purposes this can be broken down to, BLNK PV PT=(1"'TR") TE Thus each raw data point needs only to be divided by a constant and added to another constant in order to Obtain the transmittance. Absorbance is then calculated by taking the negative logarithm of the PT value. CHAPTER IV TESTING AND CALIBRATION OF THE AUTOMATED STOPPED-FLOW SPECTROPHOTOMETER The testing and calibration of the automated stopped- flow spectrophotometer is a very important part of this work. It is imperative that the accuracy, reproducibility and limits of an instrument be known in order to use it in meaningful quantitative studies. The flow system and the detection system were both thoroughly tested and characterized. The specifications of all the instruments used to test the system as well as the specifications of the components of the stopped-flow system are given in Appendix A. Only the specifications which are pertinent to this work are given. Complete specifications can be obtained from the manufacturers. A. Testing and Calibration of the Flow System When the stopped-flow drive syringes are full, they hold 2.35 ml each or a total of 4.7 ml. The amount of solution delivered per push can be adjusted by moving the stop block as explained earlier. The Optimum amount of solution to be used per push is the minimum volume I which will insure complete purging of the old solution from the Observation cell. This can most easily be determined by using a_relatively slow reaction which 63 64 results in a fairly large absorbance Change. By starting with a small push volume and increasing it until the initial absorbance starts at zero, the volume for complete purging can be found. That point was found at approxi- mately seven pushes per syringe filling. The instrument is normally run at six pushes per filling, allowing a safety margin. At this setting 0.39 ml Of each solution or 0.78 ml total are used during each push. The volume from the mixer entrance to the exit of the Observation cell is 0.16 ml. Thus, it takes approximately four times this volume for thorough purging. This factor will vary somewhat depending on the design of the flow system. Two solutions were made up to calibrate the pathlength Of the observation cell and to determine if equal volumes are delivered by the drive syringes. The solutions were: (A) 0.0017 M KZCr207, 0.2 MHZSO4 (B) 0.2 M H2804 The absorbance Of (A) and the absorbance of a 50-50 mixture of (A) and (B) were measured in a Cary 17 spectro- photometer with a standard quartz one centimeter cell, and in the stopped-flow. The pathlength of the stopped- flow was determined by comparing the absorbance values at 435 nm. The path length was calculated to be 1.94:0.01 cm, using either solution. In order to ascertain that equal volumes are delivered by the drive syringes, solution (A) was loaded into one 65 drive syringe and solution (B) into the other. The result- ant absorbance was compared to the absorbance Obtained when the solutions were switched. The values agreed with each other to within 0.15%, and in either case the absor- bance data had a relative standard deviation of less than 0.8%. This indicates that the volumes delivered by the syringes are equal, within a fraction of one percent. The dead time of the stopped-flow mixing system was determined directly by measuring the flow velocity and indirectly by the reaction extrapolation technique (24) mentioned earlier. A flow velocity transducer utilizing an opto-interruptor module was used to measure the dead time. The result was 5.08:0.09 ms versus 5.4:0.4 ms when the reaction extrapolation technique was used (112). The slightly higher dead time measured by the extrapolation technique could indicate incomplete mixing. The mixing efficiency was measured by monitoring a very fast reaction before and after stopping the flow. The reaction must be complete (assuming instantaneous mixing) in a time interval which is shorter than the dead time of the stopped-flow. A suitable reaction for this purpose is p-Nitrophenol plus sodium hydroxide, which goes to completion in less than 1 ms. Figure 5 shows the absorbance versus time curve. The beginning of the transition interval is the point where the flow stOps. It can be seen that mixing is 98-99% complete by the time the solution reaches the observation cell. However, Absorbance 66 .l89I- 0'88- .l87L- *L— Flow StOpped .l86- 0.01 M p-nitrophenol + 0.01 M NaOH* .l85 J l O 25 50 TIME (ms) Figure 5. Mixing Efficiency. *The p-nitrophenol should be in slight excess to ensure proper indication of the degree of mixing. 67 the time for the absorbance to level off seems inordinately long, another 10-20 milliseconds. This did not cause any problems with the measurements on the reactions used in this work. However, this artifact should be investigated further and corrected if possible to enable precise mea- surements on faster reactions. This artifact is apparently a mixing problem. Nevertheless the transition period remained unchanged as the air pressure was changed from 70 psi to 90 psi. B. Testing and Calibration of the Detection System The active components of the detection system are a light source, a photomultiplier tube (PMT) and its power supply, a current amplifier and the associated DAC offset circuit, and finally the analog-to-digital converter (ADC). These components are illustrated in Figures 1 and 2. The light source was easily identified as the stability limiting component of the detection system. This is in- dicated by the manufacturer's specifications (see Appendix A) and was verified experimentally. The GCA/McPherson EU-701-50 light source module used in this work has a deuterium lamp for UV work and a stabilized tungsten lamp for the visible region. In addition, the tungsten lamp has two modes of regulation, a voltage control mode and an intensity control (Optical feedback) mode. Al- though the deuterium lamp was not used in this work its stability was checked also. All intensity measurements 68 were made at 450 nm. A Heath SR-255B strip chart recorder was used for recording the spectrOphotometer output. The deuterium lamp produced drifts (over any 2 hour period) of 10% during the first 12 hours of warmup. After this warmup period maximum drifts of 1% over any 2 hour period were observed. In the constant voltage mode the tungsten lamp produced drifts on the order of 5-10% during the first two hours of warmup. After this period the maximum drift in any 2 hour period was 2%. However, the drift could be as much as 1% in a 20 minute period. The tungsten lamp gave better results when used in the Optical feedback mode. Drifts were on the order of 5-10% during the first 4 hours of warmup. After 4 hours the maximum drift in any 2 hour period was down to 1%, and after 1 day of warmup it was 0.5%. The drifts after warmup are characterized by slow changes followed by abrupt feedback corrections. These feedback corrections are the most critical in that a change on the order of the maximum drift can occur in a few seconds. The photocurrent measuring section of the detection system was thoroughly tested by determining the accuracy of the components and then determining the accuracy of the section as a unit. The section consists of the 10- bit digital-to-analog converter (DAC)-operational ampli- fier combination, the Keithley 427 current amplifier and the 12-bit analog-to-digital converter (ADC). The test 69 equipment consisted of a Power Designs 2005 precision power (voltage) source, an esi PVB 300 voltmeter bridge, a Fluke 8600A digital multimeter and a Keithley 261 precision current source. The accuracy Of absorbance measurements does not de- pend on the absolute accuracy of any of the components but only on their linearity. The linearity results re- ported here are the worst case values. The offset circuit had a linearity of 0.2% for DAC settings as low as 20 (octal). This value improved to 0.1% for settings above 200 (octal). These values are 4 10 valid for gain settings of 10 to 10 volts per ampere. For settings of 106 to 1010 volts per ampere, the linearity of the current amplifier was 0.02%. On the 105 volts per ampere setting it was found to be 0.1%. The linearity of the ADC was measured as 0.2% or :1 LSB over its entire range. The overall accuracy of the photocurrent measuring unit was determined by supplying currents from the precision current source. Simulated spectrophotometric measure- 8 ments were made by using currents of 8.99 x 10- to 8.99 x 10‘5 amperes as blank photocurrents and having the com— puter calculate absorbance values for currents which were a fraction of the blank value. The values computed from the simulated photocurrents were then compared to the theoretical absorbance values. For absorbance values from 0.0005 to 0.01, the absolute error was less than 70 0.0002. The relative error was less than 10% for absor- bances as low as 0.0005. These error values apply to both the scale expansion mode and the manual Offset mode. Above an absorbance of 0.01, the manual offset mode gave superior accuracy. For absorbance values between 0.01 and 2.25 errors were less than 0.3%. Although no improvement in the absolute absorbance accuracy is gained by using the scale expansion mode, the accuracy of reaction rate measurements may be significantly improved. In the case of small absorbance changes, resolu- tion becomes the limiting accuracy factor. By using the scale expansion mode, the resolution of the light inten- sity measurement can be improved from 1 part in 4000 to 1 part in 200,000 with better than 1% absolute accuracy. C. Accuracy of Monitoring a Chemical Reaction A well behaved chemical reaction was utilized to examine the overall reproducibility of the automated stopped-flow system. The reaction chosen for this purpose was the iron-thiocyanate reaction (113,114). Under the condi- tions used, the appropriate equilibrium is 1‘1 + SCN‘ I Fe¢ an m.m+ mmoo.o+ mwoo.o Homo.o momm.o oom.o oow.o v.wI mmoo.OI «moo.o mhvo.o mmo.a omH.o oo~.o o.~I mooo.oI mooo.o vmmo.o OHH.H omo.o omo.o Houum w Houum .>mo .Oum AomcwEuwumov so +m 30H +3 swam sm+mu< z deOAHMHusmosoo .ucmfisuumcH mm may no momusood .m manna 82 to form 12-MPA. Also the 12-MPA reaction-rate is highly dependent on acid concentration. Therefore the consump- tion of protons by Mo(VI) may affect the reaction rate indirectly. The bound acid-to-molybdate ratio was measured in nitric and sulfuric acids. Because of the limitations of the pH electrode, the maximum acid concentration used was about 0.4 molar. The results in nitric acid are shown in Table 6. The 9 coefficients were calculated, using the acid solutions with no molybdate. The results indicate that Z increases with the free acid-to-molybdate ratio and with the acid concentration. A maximum Z value of about 2.5 supports the existence of a protonated dimer, HM°206° It is unfortunate that higher acid concentra- tions could not be utilized in order to see if Z levels off at a value of 2.5. A Z value of 2.0 indicates the formation of molybdenum trioxide. Thus the Z values of 2.37 and 1.65 indicate the presence of molybdenum trioxide and even anionic molybdenum species. This contention is supported by the Observation that a white precipitate (presumably molybdenum trioxide) forms over a period of time in solutions when the free acid-to-molybdate ratio is less than about 10. Calculating Z values in sulfuric acid medium is not as simple as in the case of nitric acid because of the buffering effect of the bisulfate ion. Concentration dissociation constants, Kc' for the bisulfate ion, at 83 Table 6. Mo(VI) in Nitric Acid. Conc HNo3 Mo(VI) ga A[H+]a Std. Dev. zb 0.060 0.02 1.118 0.0330 0.0006 1.65 0.200 0.02 1.025 0.0464 0.0030 2.32 0.400 0.04 0.976 0.1012 0.0032 2.53 aAverages of 6 results. bBound acid to molybdate ratio, A[H+]/[M0(VI)] 84 various sulfuric acid concentrations, are calculated by measuring the hydrogen ion concentrations. The results in sulfuric acid medium are shown in Table 7. The Z values are slightly lower than in the nitric acid case. However, they agree within experimental error. In either acid only Z values measured after about two days were used. It was observed that Z increased to a maximum and then settled back down to a stable value within one to two days. The maximum value was as much as 15% greater than the equilibrium value. However, because Of the limited precision of the pH instrument, the relation- ship between this phenomenon and concentrations was not determined. The stable period was observed to last at least two weeks. However, after one month the solutions (especially those with low Z values) showed signs of deterioration. The pH measurements of the nitric acid solutions were made under approximately the same conditions as the de- termination Of the instrument's accuracy which was shown to be better than 5%. However, in the case Of sulfuric acid, g coefficients cannot be determined because of the uncertainty as to the exact hydrogen ion concentra- tion. The coefficients from the nitric acid solutions Of similar hydrogen ion concentrations were used. In adjusting the ionic strength of the sulfuric acid solutions to 1.00, the approximation was made that sul- furic acid in solution exists entirely as hydrogen ions 85 .ox mcflms an soHumHusmosoo sofl somouomn omusmmos may Eoum omumasoamom .usmumsoo sOHuoflOOmmwo COAumuusoocoo mummasmwm u OM p .Houomm m 0:» mafia: an msflommu Honda mm on» Sony OmswEnmumoo .muasmmu O HO momn0>€n .Am manuav sumo Owom venues on» Scum noxmem m~.~ memo.o osoo.o e~mm.o sooo.o neoe.o osm.o eo.o «He.o ~H.N eso.o mmoo.o moa~.o smoo.o mmmm.o mmo.a ~o.o so~.o ms.a eso.o omoo.o mooo.o mmoo.o Homo.o mHH.H mo.o moo.o N ox .>mo .oum AH>Voz+ .>mo .oum eomwm o AH>Oos eommm 0 O m o N 2 ecowumuucmonou on m 0.3 eaoflumnusmosoo cow somouomm .oeoa oeusmasm ea AH>Ooz .e oases 86 and bisulfate ions. The results for the reference sul- furic acid solutions indicate ionic strengths of 1.04 to 1.08. The ionic strengths of the sulfuric acid solutions containing Mo(VI) were higher by an additional 1 to 2%. As a check on the values obtained for the bisulfate concentration dissociation constants, a comparison can be made to the thermodynamic dissociation constant, K = 0.012 (119). + 2- H 80 + 2— + 2— K=E ][ 4] H — - c «— H804 y 7 From the measurements made here, |~ Y~g~l SO, KC=(-Y——2)K The ratio of the activity coefficients is determined to be 4.2 by applying the Debye-Huckel equation with ion size parameters of 3 and 4 angstroms for the bisulfate and sulfate ions, respectively. This results in a value of 0.05 for the concentration dissociation constant which is in fair agreement with the values determined in this work (Table 7). 87 B. Spectra and Properties of Molybdenyl and lZ-MPA Solu- tions Both molybdenyl and lZ-MPA solutions have an appre- ciable absorbance in the ultraviolet region of the spectrum but absorb to a much lesser extent in the visible region. The absorbance of lZ-MPA extends slightly farther into the visible region than the absorbance of the molybdenyl species does. It is in this region (above 400 nm) that 12-MPA abosrbance measurements are made when it is desir- able to minimize the blank absorbance due to molybdenyl ions. The spectra Of molybdenyl solutions from 410 nm to 430 nm were recorded in order to determine blank correc- tions for the 12-MPA absorbance measurements. The Cary 17 spectrophotometer was used to record all spectra in this work. The molar absorptivity of the molybdenyl ion was found to be a function of the free acid-to-molybdate ratio, R, rather than the acid concentration alone. Some ap- proximate values were measured at a Mo(VI) concentration of 0.04 M. They are shown in Table 8. In all cases where lZ-MPA absorbance values are reported, the appropriate molybdenyl blank absorbance has been subtracted. Molybdenyl solutions were always allowed to stand for two days or longer before use. This is necessary in order to allow the solutions time to stabilize. This 88 Table 8. Molar Absorptivity of Mo(VI)a Rb Wavelength= Wavelength= Wavelength= 410 nm 420 nm 430 nm 5-10 0.30 0.18 0.08 20-25 0.10 0.05 0.02 30.04 M Mo(VI) at an ionic strength of 3.00 (NaNO3) bFree acid-to-molybdate ratio. 89 stabilization period has been recognized by other workers in its effect on the formation of 12-MPA (120). It has been Observed directly in this work by the changing value of the bound acid-to—molybdate ratio as mentioned pre- viously. The overall equilibrium for the formation Of 12-MPA can be represented as follows: + xMO(VI) + H3PO4 4— 12-MPA + yH+ As of this writing the exact form of the Mo(VI) species has not been conclusively determined. However, evidence from this work and results from other workers (89) indicate that a protonated dimer (HMOZOE) is likely. The stoichio- metric coefficient, x, would then be 6. A value of 9 has been suggested for y, the stoichiometric coefficient of the hydrogen ion (89). A high value is in agreement with the large inverse dependence of the reaction rate on hydrogen ion concentration. This will be discussed further in the next section. The concentration effects on the equilibrium must be considered in the determination of the molar absorptivity of lZ-MPA. The molar absorptivity of 12-MPA was determined by using an excess of molybdenyl ion. The amount of excess at a fixed acid concentration is limited because as the free acid-to-molybdate ratio is lowered to about 5, molybdenum trioxide begins to precipitate. And increasing 90 the acid concentration forces the equilibrium in the re- verse direction. Using an excess of phosphate would seem to be an alternate method of forcing the reaction to com- pletion. However, if the molybdenyl ion is not maintained in excess (perhaps as much as 40 times the stoichiometric amount), 12-MPA is not the sole product. Unsaturated heteropoly compounds are formed under these conditions. This was observed visually by the disappearance of the initially formed yellow color as additional phosphate was added to a phosphate plus molybdate solution. When the phosphate concentration had reached about one twentieth of the molybdenyl concentration, the yellow color had visually disappeared. ’ The change in absorbance of a 0.1 millimolar solution of phosphate as the Mo(VI) concentration is increased is illustrated in Figure 6. As the Mo(VI) concentration is increased, the absorbance should level off, correspond- ing to a lZ-MPA concentration of 0.1 millimolar. Because Of the precipitation of molybdenum trioxide, the Mo(VI) concentration was limited to about a 50-fold excess over the stoichiometric amount. However, the absorbance curve is fairly well leveled off at that point and the limit can be estimated. The estimated molar absorptivity at I 420 nm is 770 51-molm1-cm"1 with an estimated accuracy of :70. Spectra of lZ-MPA solutions were recorded from 410 nm to 430 nm in order to determine the change in molar Absorbance at 420 nm 91 .4353? E£ HH—lH—I .06- 004 - .OZr z .00 1 4 i: 4___|__n .00 .0! .02 .03 .04 .05 .06 [Mo(VI)] (moles/liter) Figure 6. Molar Absorptivity of 12-MPA. - stopped-flow data, normalized to 1 cm path length - Cary 17 data, 1 cm cell [H3PO4] = 1.00 x 10-4 M [3+] = 0.0400 M (nitric acid) 92 absorptivity with wavelength. The results are given in Table 9. C. Kinetics of the Formation of 12-MPA in Nitric Acid Solutions The latest published study of the kinetics of formation of lZ-MPA proposed a complicated rate law involving acid concentration to the inverse eighth, inverse fourth, in- verse second and zeroth order for both nitric and per- chloric acid (121). A slightly different rate law was determined for sulfuric acid solutions. In all cases acid was added only to the molybdenum(VI) solutions be- fore mixing. Although this procedure maintains a constant free acid-to-molybdate ratio, R, it has the disadvantage that the acid concentration changes upon mixing. Since the form of the molybdate species depends on R, rather than the acid concentration pgg'gg, that procedure would eliminate effects on the rate of formation of 12-MPA due to transformation of the Mo(VI) reactant. However, the change in acid concentration can cause a temperature change due to the heat of dilution. This is significant for sulfuric acid at higher concentrations. For example, at a final acid concentration of l M, there would be 0.7°C increase in temperature upon mixing for sulfuric acid (122). The change for nitric acid or perchloric acid would be less than 0.01°C (123). 93 Table 9. Molar Absorptivity of lZ-MPA Solutionsa Wavelength, nm 410 420 430 Relative Absorbanceb 1.00 0.72710. 009 0.514:0.008 Molar Absorptivityc, l'mOl-lcm-l 1060:100 770170 540:50 annic strength is 3.00 (NaNO3) bAverage of 6, the absorbance at 410 nm is the basis. cBased on the measured value at 420 nm (Figure 6), with an estimated standard deviation of 70. 94 In this thesis work, the initial reaction rate was found to increase by a factor of 1.98 i 0.10 for a change in temperature from 21.0 to 24.8°C. This was unchanged for nitric acid concentrations ranging from 0.2 to 0.4 M. This corresponds to an Arrhenius activation energy of 31.3 i2.3 Kcal/mole. Beckwith and coworkers found the activa- tion energy to be approximately the same (21 Kcal/mol) in all three acids (121). Although the determination in this present work involved only two temperature levels, the temperature measurements were quite precise (t0.02°C) and the thermistor was immersed in the reacting solution. In the other study, the temperature Of the thermostating water was the measured quantity. Applying these results to the change in temperature by acid dilution (0.7°C) gives a change in reaction rate of 9% or 13% depending on which value for activation energy is used. An experiment was carried out to compare the rates of reaction when all the acid was in the Mo(VI) solution before mixing and when the acid was evenly distributed between the Mo(VI) solutioniand the phosphate solution. The acid concentration after mixing was 0.4 M in either case. The average rates from the two different procedures were identical within experimental error. For the acid dependence study, solutions were made up at constant phosphate and Mo(VI) concentrations, and the nitric acid concentration was varied to determine the dependence. In adjusting the acid concentration, it 95 was assumed that 28 protons were consumed per Mo(VI) atom. The ionic strength was adjusted to a constant value with sodium nitrate. Reagent Grade or Analytical Reagent Grade chemicals were used in all cases. Concentrations within a series were varied by dilution of one stock solution. In all determinations, the absorbance versus time curve used to calculate the initial reaction-rate was the point by point average of eight runs. The rate was determined as the linear least squares slope of a selected portion of the absorbance curve (124). The method of selecting this portion is illustrated in Figure 7. A Savitzky-Golay first derivative smoothing routine (125) was used to create a rate curve. Both this rate curve and the original absorbance curve are shown in the figure. The curves were displayed via the CRT terminal for visual discrimination. The plateau of maximum rate is the selected region which is then used to calculate the initial rate by the linear least squares fit of the absorbance data to a straight line. The standard deviations listed in this study are the calculated standard deviations of the slope of the best line through the selected points. This value may be different from the standard deviation obtained I by determining slopes for individual runs and then cal- culating the standard deviation of the set of slopes. The rate data are given in Tables lO-A, lO-B and 11. Table lO-B contains data for solutions with R values of 96 (puooas/aoueqlosqe) aqeu .soauoeom «azumfl 0.0 .mosmnHOmnm mo 0>Huw>aumo umuflm on» was mononuomnfl .h musmflm Amocoommv mafia 0.0 0.0 00. .0... N0.t n0.l sot no. I. . . . _ 1 q m 4 n 00. ........... :3 u eoamm .... l. . ..:... No.0 "THC/Com: .. no 6.... s; I. :9 1 mm ..... ... l loo. T 0.0... on... Otnuuu. .. 0. 1| coo... coo... to. J ........ .....:.:.... L N _. fl 0000.000. coo-oo- Lm—o aoueqxosqv 97 Table 10-A. Initial Rate of Formation of lZ-MPA, Conditions I Conditions: 24.810.2°C [Mo(VI)] = 0.01 M [H3PO4] = 5 x 10'5 M Ionic Strength (NaNO3) = 2.0 Initial Reaction [HNO3], Time Rate.b M Interval, sa A/s x 103 Std. Dev. x 104 0.22 0.85-1.81 8.095 0.22 0.79-1.63 8.071 2.053 0.25 0.79-1.87 7.347 1.386 0.25 0.97-1.87 7.348 1.646 0.30 0.97-1.87 6.165 2.042 0.30 0.41-1.91 6.056 2.020 0.30 0.85-1.69 5.824 0.460 0.35 0.91-1.99 4.997 1.340 0.35 0.85-1.99 5.067 1.434 0.40 1.21-2.83 3.984 0.863 0.40 1.21-2.41 4.030 1.239 0.45 1.21-2.17 3.348 1.727 0.45 0.85-2.35 3.241 0.970 0.50 1.03-3.13 2.520 0.585 0.50 1.63-3.01 2.485 1.109 0.55 1.15-3.55 1.771 0.461 0.55 1.45-3.91 1.751 0.431 0.60 1.93-5.29 1.066 0.284 0.60 1.15-3.97 1.056 0.334 0.60 1.57-4.09 1.048 0.244, 0.60 1.63-4.15 1.121 0.180 0.70 1.82-4.42 0.359 0.177 1.781 aReaction time interval during which the rate was measured. bThe rate in mol-i'l-sec'1 can be calculated by using a path length of 1.94 cm and a molar absorptivity of 7701702' mol-locm'1 at 420 nm. Table lO-B. Initial Rate of Formation of 12-MPA, Condi- tions I. Conditions: Same as Table 10-A Initial Reaction [HNO3], Time a Rateb 3 4 M Interval, s A/s x 10 Std. Dev.)c10 0.16 0.97-1.87 8.254 1.908 0.16 0.79-1.63 8.115 2.111 0.16 0.85-1.75 8.074 2.118 0.16 0.88-1.78 8.625 1.531 0.18 0.85-1.81 8.333 1.828 0.18 0.97-1.81 8.274 1.868 0.20 0.67-1.81 8.295 1.309 0.20 0.67-1.75 8.260 1.457 aReaction time interval during which the rate was measured. b The rate in mol-z- 1 -sec"1 can be calculated by using a path length of 1.94 cm and a molar absorptivity of 7703702- mol‘locm“1 at 420 nm. 99 20 or less. In Table 11 the acid concentration of 0.20 which results in an R value of 10 is not listed separately because of the shortness of the table. The reason for differentiating solutions with R values of less than about 20 is the apparent formation of unreactive Mo(VI) species. This formation results in the leveling Off of the rate curve at low acid concentrations. Several observations support the contention that this "zero order" region is caused by unreactive Mo(VI) species rather than a real zero order dependence on acid concentration. First, the pH results discussed previously indicate the presence of MOO3 or even anionic Mo(VI) species at low R values. Second, it was Observed that those solutions form a precipitate of MOO3 over a period of time. This precipitation was Ob- served to be hastened when the solution was placed in a glass container (normally the solutions are kept in poly- ethylene bottles). And finally, it was observed that the best-fit rate equations containing zero order terms (in acid concentration) actually "ignored" the "zero order" region of the data and leveled off at much lower acid con- centrations. A plot of the data in Tables lO-A and lO-B and the curve generated by the best-fit equation is shown in Figure 8. All data were analyzed using KINFIT, a general purpose curve-fitting and equation solving program (115). Since the data with acid concentrations of 0.20 M and below were not used in the curve fit, the best fit curve is terminated at an acid concentration of 0.22 M. 100 Table 11. Initial Rate of Formation of lZ-MPA, Conditions II. Conditions: 24.8:0.2°c [Mo(VI)]=0.02 M [H3PO4]= 10‘4 M Ionic Strength (NaNO3)=3.0 Initial Reaction [HN03], Time a Rateb 2 4 M Interval, s A/SJclO Std. Dev.)(10 0.20 0.73-1.51 2.370 1.926 0.20 0.73-1.51 2.358 2.228 0.40 0.73-1.57 2.257 2.607 0.40 0.73-1.57 2.245 1.763 0.80 1.45—3.31 0.611 0.592 0.80 1.39-2.59 0.615 1.359 1.00 1.87-5.47 0.148 0.228 1.00 2.21-4.41 0.149 0.253 aReaction time interval during which the rate was measured. bThe rate in mol'l-l-sec-1 can be calculated by using a path length of 1.94 cm and a molar absorptivity of 770:70£° mol"l-cm"1 at 420 nm. 101 9- 8- fig ‘TL "best fit" curve 6‘ g .. s.) 6- m \e 0 O 8 f: 5r 0 m .O .2 fig ‘Ir H x V‘ m 1“ E 3' 2:. [- C) J, 1_ 1 1 1 0.I 0.2 0.3 0.4 0.5 0.6 0.7 [H+] (moles/liter) Figure 8. Dependence of the Rate of Formation of lZ-MPA on [HN03], Conditions I. 102 Based on the results from this data, it could not be said with certainty whether inverse eighth and inverse first order or inverse ninth and inverse first order gave the better fit. Other rate laws were tried but none gave a satisfactory fit. The results for the two cases are shown in Table 12. The inverse ninth order equation gave slightly better standard deviations for the conditional rate constants in all cases. However, when the data were split into a high acid set and a low acid set, the Change in the value for the rate constant on the ninth order term was disturb- ing. Further analysis showed that at the low acid concen- trations, which produced the inordinately high rate constant, there is very little dependence on the ninth order term. In fact, the large rate constant change from 56,036 to 104,963 shifts the curve an average of less than 0.8% in the low acid region. This is within the experimental error Of the data. The acid rate law is further clarified by analysis of the data in Table 11. These data are relatively more precise than the data in Tables lO-A and lO-B because of the higher concentrations of Mo(VI) and H3PO The 4. results of fitting the two rate laws to this more precise data are given in Table 13. The best fit rate curve along with the data are plotted in Figure 9. The data at an acid concentration of 0.20 M were not included in the fit because of the low R value. There is little doubt that the better fit rate law involves acid concentration 103 Table 12. Determination of Rate Constants, Conditions I. Range of Rate Concentration , Ma Equationb Kl RSD , % K 2 RSD , % 0.22-0.70 A 34,440 2.3 554.6 0.82 0.22-0.70 B 57,539 2.1 562.8 0.68 0.22-0.40 A 42,284 27.2 553.9 1.1 0.40-0.70 A 35,274 3.1 536.6 2.8 0.22-0.40 B 104,963 21.7 555.7 0.79 0.40-0.70 B 56,036 2.6 579.2 2.0 aSee Table 10-A for data quuation A: Rate 1/K1[H+]8 + K2[H+], absorbance/second Equation B: Rate l/K1[H+]9 + K2[H+], absorbance/second. 104 Table 13. Determination of Rate Constants, Conditions II. Range of Rate Concentration, Ma Equation K 1 RSD, % K2 RSD, % 0.40-1.00 A 526.9 4.6 1.09.2 2.3 0.40—1.00 B 560.2 0.43 110.7 0.20 aSee Table 11 for data 1/K1[H 1/K1[H+]9 + K2[H+], absorbance/second. b +]8 + K2[H+], absorbance/second. Equation A: Rate Equation B: Rate .HH mcofluwwcoo .flmozmu co észNH mo aoflumEuom mo wumm any mo mocmccmawo Anmuwa\mmaoev m .m musmflm 0.. 0.0 0.0 «.0 N0 . .04 .|qw 1: 105 m>uso =0Hm ummn= L V.N (puooes/eoueqxosqe) ZOT x 3323 106 to the inverse ninth order. Thus this work indicates that the rate law in terms of acid concentration is 1 Rate = K1[H+]g+ K2[H+] , absorbance/second where K1 and K2 are conditional rate constants which de- pend on [Mo(VI)] and [H3PO4]. Other workers have shown that the rate is dependent on [H3P04] to the first power but the dependence on [Mo(VI)] is more complicated (89). Although the dependence on Mo(VI) concentration was not determined in this work, some limiting conditions were. As mentioned previously in this work, concentration of Mo(VI) must be greater than about forty times the concen- tration of H3Po4 in order to avoid the formation of un- saturated heteropoly compounds. Evidence for this conten- tion is also given in the next chapter. Another restraint on the Mo(VI) concentration is imposed by the requirement of keeping the free acid-to-molybdate ratio, R, above 20 to avoid the formation of unreactive Mo(VI) species. Thus for a given phosphate concentration and a given acid con- centration, the first restraint will determine the upper limit for Mo(VI) concentration and the second restraint will determine the lower limit. In analyzing rate data at low acid concentrations, an apparent transformation of lZ-MPA was observed. This would be analogous to the transformations observed in similar heterOpoly compounds of Mo(VI) (99,100,126-130). This 107 transformation of lZ-MPA was observed to be dependent on the acid concentration per se and not on R. The trans- formation was observed for all acid concentrations between 0.16 and 0.25 M, but never for concentrations of 0.30 M and above. At an acid concentration of 0.25 M, the trans- formation was observed with R equal to 25 whereas at an acid concentration of 0.40 there was no measurable trans- formation for R values between 10 and 40. The rate of transformation was too slow in most cases to be measured quantitatively but there was no doubt about its presence at low acid concentrations. The transformation was observed as a slow (several orders of magnitude slower than the initial rate) increase in absorbance which was detectable long after the forma- tion of lZ-MPA should have been complete. The point at which equilibrium should be attained can be estimated by observing a reaction of higher acid concentration in which the equilibration is attained without transformation. The primary equilibration is slower at higher acid concen- trations so that the estimate of the equilibration point is conservative. The transformation has been measured for periods over five times as long as the estimated equilibration time and in that time (20 to 100 seconds) the absorbance was measured to increase up to 12.811.0%. It should be noted that acid concentrations below 0.30 M would generally not be used for analytical procedures and thus this transformation would not be observed. CHAPTER VI THE REACTION-RATE ANALYSIS OF PHOSPHATE AND SILICATE The reaction-rate analysis of phosphate "unknowns" was performed under the control of the PDP 8/e minicom- puter. The experimenter had only to specify the portion of the reaction curve to be used to determine the initial rate. A working curve was also developed for silicate analysis. The reaction of silicate with Mo(VI) at high acid concentrations is similar to the phosphate reaction, but proceeds at a much slower rate (99,100,126-130). Reaction-rate data on standards and unknowns are pro- cessed by the computer and the analytical results printed out. The reaction-rates and concentrations of the standard solutions are fit to a linear equation (124). That equa- tion is then used to determine the concentrations of the unknown solutions from their reaction rates. The com- puter programs are set up to handle up to 20 standards and 50 unknowns including averaging of multiple runs of each solution. This capability can be easily expanded to handle a greater number of solutions. The experiments in this chapter were accomplished prior to the chemical studies discussed in Chapter V so that the limiting conditions determined in that work were not utilized here. In all experiments in the present chapter, the acid was contained only in the Mo(VI) solution before 108 109 mixing. However, since nitric acid was used, there was no problem with temperature effects due to heats of dilu- tion. The acid concentrations given are the concentrations after mixing and no corrections were made for the amount of acid consumed by Mo(VI). Although optimum conditions were not used in these analyses, the results are indica- tive of what might be expected from the reaction-rate analysis of phosphate and silicate via the formation of their respective heteropolymolybdates. Table 14 shows the computer printout of the results of the analysis of phosphate "unknowns". Only the column containing the actual concentrations of the "unknowns" was added to the original printout. The slope and inter- cept of the linear equation are printed out along with the data and results. The relative accuracy is 12% at 0.2 ppm P (as H3PO4) and improves to better than 2% for the concentrations from 0.5 to 3.5 ppm P. If only the data for the highest and the lowest standard concentrations are used to determine the working curve, the errors range from 4.5% at 0.2 ppm P to 1.9% at 3.5 ppm P. However bend off of the analytical curve occurred at concentra- tions of 7 ppm and above. This is illustrated in Figure 10. The solid line represents the best linear fit for ' all concentrations up to 4 ppm, and the dashed line represents the best linear fit for all concentrations up to 20 ppm. It can be seen that the latter case results in extreme relative errors at low concentrations. As Table 14. 110 Reaction-Rate Analysis of Phosphate. Std. Conc.b Slope (Absorb/sec)a Std. Dev. of Slope 0.2000E+00 0.1059E-02 0.8213E-04 0.1000E+01 0.5233E-02 0.8126E-04 0.2000E+01 0.1033E-01 0.8437E-04 0.400E+01 0.2034E-01 0.9103E-04 Initial Rate = A * Concentration + B A = 0.5066E-02 B = 0.1232E-03 Unk. Slope Std. Dev. Actualb'c No. Conc. (Absorb/sec)a of Slope Conc. l 0.1756E+00 0.1013E-02 0.7849E-04 0.200 2 0.4977E+00 0.2645E-02 0.8585E-04 0.500 3 0.2033E+Ol 0.1042E-01 0.8334E-04 2.00 4 0.3557E+01 0.1814E-01 0.8612E-04 3.50 A b Concentrations in ppm P, lppm P = 3.23x10- 5 Rate of formation of 12-MPA; [Mo(VI)] = 0.080M,[HNO3]=0.64M. M H3PO4. cThis column not included in original computer printout. 111 L. .08 / 007'. / 8 § 006‘" / 8 / g / 0.080 M Mo(VI) g .05 0.64 M HNO3 O 3 3 3 .04} g 3 .03 4.) '5 .02 .O|< . 0 1 l 1 1 O 0 5 IO I5 20 Concentration (ppm P) Figure 10. Reaction Rate Analysis of Phosphate, Low [Mo(IV)]. Initial rates measured from 0.6 to 1.4 seconds. Solid line represents best linear fit of points from 0.2 ppm to 4.0 ppm. Dashed line represents best linear fit of all points. 112 discussed previously in this thesis, the reason for the bend off of the curve is the formation of unsaturated hetero- poly products at low Mo(VI) to phosphate ratios. From Figure 10, it can be estimated that the mole ratio of Mo(VI) to phosphate should be at least 350 (30 times the stoichiometric ratio) to prevent the formation of un- saturated products. The same range of phosphate concen- trations was used with a higher Mo(VI) concentration to demonstrate this point. The reaction-rate results are listed in Table 15 and plotted in Figure 11. The improve- ment in linearity is marked. Fairly good linearity is obtained over two orders of magnitude. Reaction-rate data were obtained for the formation of the lZ-molybdosilicate anion (lZ-MSA) under the same condi- tions as used for the formation of lZ-MPA. The results are given in Table 16 and plotted in Figure 12. The results are similar to those for lZ-MPA, but relatively less precise because of the slower reaction-rate. The non-zero intercept indicates some bend off in the analytical curve. This is attributed to an insufficient excess of Mo(VI) and thus the formation of unsaturated heteropoly products at the higher silicate concentrations. All rates listed were the average of eight runs. 113 a Table 15. Reaction-Rate Data: Phosphate Analysis b Time Interval Conc. (sec) Slope Std. Dev. (Absorb/sec)C of Slope 0.8900E+00 0.20 0.5000E+00 to 0.1692E-02 0.1863E-03 0.8900E+00 1.00 0.5000E+00 to 0.7120E-02 0.2096E-03 0.8900E+00 4.00 0.5000E+00 to 0.2826E-01 0.1902E-03 0.8900E+00 10.0 0.5000E+00 to 0.6896E 01 0.2129E-03 0.8900E+00 20.0 0.5000E+00 to 0.1361E+00 0.2239E-03 0.8900E+00 a[Mo(VI)] = 0.176, [HNO3] = 0.64 M b Parts per million phosphorous. cRate of formation of lZ-MPA. .I4 .IZ .IO .08 .06 .04 Initial Rate (absorbance/second) .02 .00 0 Figure 11. A 0 Initial rates 114 0.176 M Mo(VI) 0.64 M HNO3 1 L 1 4 5 10 I 5 20 Concentration (ppm P) nalytical curve for Reaction-Rate Analysis f Phosphate. measured from 0.50 to 0.89 seconds. 115 Table 16. Reaction—Rate Data: Silicate Analysis b Time Interval SlOpe Std. Dev. Conc. (sec) (Absorb/sec)C of Slope 2.0 0.4100E+00 to 0.5403E-02 0.16613-04 0.3410E+01 4.0 0.4100E+00 to 0.1175E-01 0.1982E-04 0.3410E+01 8.0 0.4100E+00 to 0.2123E-01 0.2210E-04 0.3410E+01 16.0 0.4100E+00 to 0.4149E-01 0.4318E-04 0.3410E+01 aRate of formation of lZ-MSA: [Mo(VI)] = 0.176 M, [HNO3] = 0.64 M bppm Si, 1 ppm Si = 3.56 x 10"5 M HZSiO3. 116 004'. oCfiS" 0.176 M Mo(VI) 0.64 M HNO3 1&)2.- Initial Rate (absorbance/second) 0C”?- l l l ._J “000 4 8 12 IS Concentration (ppm Si) Figure 12. Analytical curve for the Reaction-Rate Analysis of Silicate. Initial Rates measured from 4 to 34 seconds. CHAPTER VII FUTURE PROSPECTS A. The Automated Stopped-flow Instrument Accurate analysis, high sample throughput and automa- tion are major considerations in the design of analytical instruments and in the development of analytical tech- niques. The stopped—flow technique offers the potential for rapid accurate analysis and is readily amenable to automation. This present work was undertaken with those considerations in mind. In this thesis work, the stopped-flow mixing system was completely thermostated and a thermistor was inserted into the flow channel for accurate solution temperature monitoring on the millisecond time scale. A new mixer was installed which improved the mixing efficiency. In addi- tion, optics were designed to permit much greater radiation throughput, and the spectrophotometric detection system was redesigned to permit high precision measurements. The observation cell was also equipped with platinum elec- trodes for conductivity measurements, although they have not been used in this present work. The stopped-flow instrument was interfaced to a PDP 8/e minicomputer so that the Operation of the stopped— flow spectrophotometer and the collection and analysis of data could be done under computer control. Extensive 117 118 computer software has been developed to allow fundamental kinetics studies and routine reaction-rate analysis. The software is quite versatile and can be easily modified to accommodate different types of analyses or changes in the instrumentation. The limiting factor affecting the accuracy of the spectro- photometric data is the fluctuation of the light source intensity. This can be eliminated as the limiting source of error by monitoring the light source intensity and correcting the spectrophotometric data for any fluctuations. This can be accomplished by inserting a beam splitter between the monochromator and the observation cell and using a separate PMT detector powered by the same supply as the main PMT. Several workers have achieved very accurate spectrophotometric results by monitoring the light source intensity (37,39). Light source intensity data can be collected by the computer along with the spectrophotometric data, and the latter corrected by software. In this present work, errors in the spectrophotometric data were introduced when temperature data was being col- lected in the same run. The absorbance data for an entire run would occasionally be reduced by a factor of about two. This is presumably a problem with the interface buffer electronics or with the electronics of the tempera- ture circuit. New connectors may have been installed in the buffer box and this may eliminate the problem. Although the new mixer has improved the mixing 119 efficiency, there is still a need for further improvement. It is desirable to eliminate or reduce the 10-20 milli- seconds it takes for the mixing to progress from 98-99% complete to 100% complete after the flow stops. This can be a problem for monitoring fast reactions. Dr. Dye and coworkers in this chemistry department have had very good results with a tangential jet double mixer (41,131). This design would probably be the most fruitful approach be- cause of the close availability of the expertise. The mixer housing was purposely designed for easy replacement of the mixer so that different designs could be readily inserted and studied. The one time limiting step remaining in the analysis procedure is the preparation of samples. An accurate (0.1%), wide ranging (6 orders of magnitude), versatile solution preparation system is presently being constructed in our laboratory (132). This system is to be controlled by a microcomputer and can be initialized by the PDP 8/e minicomputer to deliver specified solutions to the stopped- flow mixing system for analysis. The new software can be easily integrated with the existing software to enable hierachical control of the entire system for completely automated stopped-flow studies. 120 B. Study of the Formation of lZ-Molydephosphate and Re- lated Mo(VI) Compounds Mo(VI) reacts with phosphate, silicate, arsenate and germanate at high acid concentrations to form yellow heteropolymolybdate compounds. This present study was concerned with the formation of lZ-MPA and, to a much lesser extent, with the formation of lZ-MSA. In either case, it is important to know the form of the Mo(VI) species in the highly acid solutions. This work demonstrated the feas- ibility of determining (via pH meter) the number of protons consumed by the Mo(VI) species upon acidification. The results showed that up to 28 protons were consumed per Mo(VI) (R=2.5) as the free acid-to-molybdate ratio, Z, was increased to ten. This result indicates the formation of a protonated dimer, HMoZOz. This study should be carried out to higher z values. The present results indicate that meaningful data would be expected up to acid concentrations of 0.6 M, with a Mo(VI) concentration of 0.02 M. This proposed study would indicate whether a plateau for the value of R has been reached and thus whether the protonated dimer would be the dominant species in the concentration range of interest. The dependence of the rate of formation of lZ-MPA on nitric acid concentration has been studied in this work. The results indicate inverse ninth and inverse first order dependence. .These results are valid only for Z values above 10 because of the unreactivity and instability of 121 the Mo(VI) species below this value. Previous workers have determined that the rate dependence on phosphate concentration is a simple first order dependence (66,121). However, the dependence on Mo(VI) concentration was more complicated and terms in the rate law consisting of a ratio of acid concentration to Mo(VI) concentration were postu- lated. Thus it is necessary to perform further experiments in order to determine the rate dependence on Mo(VI). In this proposed study, the concentration of Mo(VI) should be maintained greater than that of phosphate by a factor of about 100 to avoid the formation of unsaturated hetero- polymolybdates. Additionally the rate laws should be determined in perchloric and sulfuric acids. The accidental discovery of the possible transformation of the lZ-MPA species is in need of further corroboration. The occurrance of this transformation appears to be de- pendent on the acid concentration per se rather than on Z. It has only been observed for acid concentrations below 0.30 M. This type of transformation has been observed for other heteropolymolybdates (99,100,126-128). In those studies, differences in spectra and reduction potentials confirmed the presence of the two distinct species. Those techniques should prove useful in the identification of the two 12-MPA species. Also, the rate of transformation between the lZ-MPA species and the molar absorptivities of the two species are in need of further investigation. The molar absorptivity of the 122 species which is formed at higher acid concentrations has been estimated in this present work to be 770 t 70 IL'mol"1 -cm"1 at 420 nm. The determination of the rate law and molar absorp- tivities would facilitate the determination of the equi— librium expression for the formation and dissociation of lZ-MPA. The rate law and derived mechanism would suggest possible equilibrium expressions which could be tested with equilibrium and kinetics data. The major goal of these fundamental studies is to specify conditions for the analysis of phosphate. The results of this present study indicate that for high stability of the Mo(VI) solutions the 2 value should be above 20. However, faster reaction-rates are achieved for somewhat lower acid concentrations. This suggests having all the nitric acid contained in the Mo(VI) solution before mixing. This would not be appropriate for sulfuric acid because of the large heat of dilution. It is also desir- able to have as high a Mo(VI) concentration as practical in order to increase the upper limit of phosphate concentra- tion without forming unsaturated heteropolymolybdates. Increasing Mo(VI) concentration would also appear to increase the reaction rate. However, in order to main-' tain a high enough Z value, the acid concentration would also have to be increased. This leads to a decrease in the reaction rate as the concentrations are increased beyond a certain point. An optimum value would be a Mo(VI) 123 concentration of about 0.3 M. At this Mo(VI) concentra- tion, the reaction-rate analysis of phosphate should be studied at acid concentrations from 0.3 M to 0.8 M. In addition to sensitivity and dynamic range, long term stability of the Mo(VI) reagent should be investigated. The results on the formation of lZ-MPA can then be used for a basis in studying the formations of lZ-MSA and the arsenate and germanate analogs. A preliminary study of all four of these species has been done by Halaz and Pungor (99,126). APPENDICES APPENDIX A INSTRUMENT AND COMPONENT SPECIFICATIONS This appendix contains manufacturers specifications of the instruments and components used in this work. Only the pertinent specifications are listed. More complete listings can be obtained from the manufacturers. The equipment is divided into three categories; stopped-flow components, stopped-flow test equipment and analytical instruments. 1. Stopped-flow Components a) Light source, Model EU-701-50 GCA/McPherson Instrument Company Deuterium Lamp Spectral range: 175-450 nm Stability: Less than 1% drift over 2 hours (after 30 minutes warm-up) Tungsten Lamp Spectral range: 350-3000 nm Stability-voltage Control: 1% over 2 hours Stability-intensity control: 0.1% for short periods, less than 1% drift over 2 hours (after 30 minutes warm-up) b) Monochromator, Model EU—700 GCA/McPherson Instru- ment Company 124 125 Accuracy 0.1 nm Wavelength range: 190-1000 nm Aperature ratio: f/6.8 @200 nm Focal length: 350 nm Stray light: less than 0.1% between 220 and 600 nm Spectral bandwidth: continuously adjustable between 0.05 and 8 nm c) Quartz flexible fiber optic bundle Schott Optical Company Length: 25 cm Diameter: 2 mm Aperature angle, 20* 32t5° @ 254 nm 55:5° @ 546 nm Transmission 50% @ 250 nm 55% @ 300 nm 59% @ 500 nm 59% @ 700 nm *a is the angle for which the transmission is 50% of the transmission at an angle of 0°. d) Quartz internally reflecting rod Schott Optical Company Length: 10 cm Diameter: 3 mm Aperature angle, 2a: 40:5° @ 254 nm 40:5° @ 546 nm 126 Transmission: 85% @ 220-1100 nm e) 1P28 Photomultiplier tube RCA corporation Luminous Sensitivity: 200 amperes per lumen 10 Dark current: 2 x 10 amperes maximum at 600 VDC f) High voltage power supply for PMT, Model EU-42A Heath Company Range: 300-1500 VDC Current: 1.5 mA, max Voltage regulation with line voltage change of 105 to 125 VAC: 0.05% of full scale With Load current change from zero to maximum: 0.1% Ripple: Less than 5 mV peak, with 1000 VDC at 1 mA output 9) Current Amplifier, Model 427 Keithley Instruments, Inc. Range: 104 to 1011 volts per ampere in eight decade ranges Output: ~10 to +10 V at up to 3 mA Output resistance: less than 10 ohms, dc to 30 KHz Output accuracy: 2% or reading to 109 V/A range Rise time (10% to 90%) Nominally adjustable from 0.01 to 300 ms 127 Voltage drift: Less than 0.005%/°C Effective input Less than 15 ohms on the 104 resistance: and 10S V/A ranges, increasing to less than 4 megaohms on the 1011 V/A range. h) lO-bit digital-to-analog converter (DAC), Model EU-800-GC - Heath Company. Voltage Mode Specifications Range: 0 V to -10 V at 5 mA Accuracy: :1 LSB (0.10%) Linearity: :fi LSB (0.05%) Settling time to 0.05% of full scale: 25 microseconds for 10 V step Temp. coef., zero: 10 ppm of full scale/°C gain: 50 ppm of reading/°C i) Operational Amplifier, Model AD518K (used in the offset circuit) Analog Devices Company Output voltage range: -12 to +12 V Output current: -10 to +10 mA Slew rate, unity gain: 50 microseconds, min. Settling time to 0.1%: 800 ns Temp. coef. of input offset voltage: 15 microvolts/°C, max. Differential input impedence: 0.5 megaohm, min. 128 j) 12-bit analog-to-digital converter (ADC), Model DAS-16-M12B Datel Company Range: -5 to +5 V Input Impedance: 100 megaohms. Acquisition time: 5 microseconds to 0.025% Aperature time: 50 ns Accuracy: 0.025% of full scale Throughput rate: 50 KHz Temp. coef.: 40 ppm/°C Stopped-flow Test Equipment a) Potentiometric voltmeter bridge, Model 300A Electro Scientific Industries (esi) Nominal accuracy: 0.01% b) Digital multimeter, Model 8600A Fluke Corporation Voltmeter Ranges: :200 mV, :Z'V, :20V, :200V, ilZOOV Resolution: 10 microvolts on 200 mV range Accuracy: 0.02% of input + 0.005% of range (for 2, 20, 200 V ranges) 15°C to 35°C: 0.02% of input + 0.008% of range for 1200V range 0.04% of input + 0.01% of range for 200 mV range DC input resistance: Greater than 1000 megaohms for 129 Common mode noise rejection: 200 mV and 2V ranges, 10 mega- ohms for 20V, 200V and 1200V ranges 120 dB minimum c) Precision power source, Model 2005 Power Designs, Inc. Accuracy: Range: Ripple and noise: Temp. coef.: 0.1% :1 mV at outputs below 10V 0-20 VDC less than 100 microvolts peak Less than 0.001%/°C. d) Precision current source, Model 261 Keithley Instruments, Inc. Worst case accuracy: Temp. coef. 4 7 to 10- A 0.25% for 10- 0.5% for 10'8 to 10'7 A 0.8% for 10"9 to 10‘8 A 7 0.01%/°C for 10- to 10"5 A ranges 0.1%/°C for 10"12 to 10'8 A ranges e) Strip chart recorder, Model SR-ZSSB Heath Company DC input ranges: Overall error: Standardization error: Zero setting drift: Balancing time: 10 mV, 100 mV, 1 V, 10 V Less than 1% of full scale Less than 0.005%/°C Less than 10 microvolts/°C, 10 mV range Less than 1 5 full scale 130 Analytical Instruments a) Cary Spectrophotometer, Model 17 Varian Instrument Company Wavelength range: 186-2650 nm Wavelength accuracy: 0.4 nm Stray light: Less than 0.0001% between 240 and 500 nm Photometric accuracy: 0.002 absorbance on 0-1 range 0.0005 absorbance on 0-0.l range Zero absorbance Less than 0.0005 absorbance per stability hour drift with standard VIS-IR source. b) Servo-digital pH/volt meter, Model EU-302 A Heath Company Range: 0-14 pH Resolution and precision 0.02 pH Accuracy: 0.5% of full scale. Temperature compensa- tion 0—100°C, manual control c) Combination pH electrode, Model 830072-15 Sargent-Welch Scientific Company Range: 0-14 pH Temperature range: 0-80°C APPENDIX B A BRIEF DESCRIPTION OF THE CAPABILITIES OF THE COMPUTER PROGRAMS The reader is referred to the "OS/8 Handbook", Digital Equipment Corporation, Maynard, MA, for clarification of the nomenclature used. I. IV. II. -INFORMATION FOR USERS OF PN.??? * PROGRAMS ----- PNSFl. PA--CHAINS WITH PNF?01.FT PNSF3. PA--CHAINS WITH PNF?03.FT PNSFl. PH: LO PNSFl 1) USES CHANNEL 0 of ADC 2) UTILIZES AUTOMATIC OFFSET-CAN OBSERVE 100% T TO, EG, 90% T FULL SCALE OF ADC 3) CALIBRATES OFFSET WITH LIGHT SOURCE SHUTTER CLOSED 4) LIGHT INTENSITY DATA IS TAKEN AS SPECIFIED BY THE USER AND STORED IN FIELD 1 (COMMON) ALONG WITH OFFSET AND AMPLIFICATION PARAMETERS 5) CHAINS TO SPECIFIED FORTRAN PROGRAM PNSF3. PA: LO PNSF3 1) USES CHANNEL 0 OF ADC 2) MANUAL OFFSET AND AMPLIFICATION-0% T AND 100% T MUST BE WITHIN THE RANGE OF THE ADC (-5V to +5v) 3) COMPUTER RECORDS 0% T LEVEL WITH THE LIGHT SOURCE SHUTTER CLOSED 4) RELATIVE LIGHT INTENSITY DATA IS TAKEN AS SPECIFIED BY THE USER AND STORED IN FIELD 1 (COMMON) ALONG WITH BLANK AND 0% T LEVELS 5) CHAINS To SPECIFIED FORTRAN PROGRAM PNSFTI. PA--CHAINS WITH PNT?01. FT PNSFT3. PA--CHAINS WITH PNT?03. FT PNSFT1.PA: LO PNSFTI 1) SAME AS PNSF1.FT EXCEPT TAKES TEMPERATURE DATA ALONG WITH THE ABSORBANCE DATA PNSFT3.PA: LO PNSFT3 131 III. IV. 132 1) SAME AS PNSF3.FT EXCEPT TAKES TEMPERATURE DATA ALONG WITH THE ABSORBANCE DATA PNF?01.FT--CHAINS WITH PNSF1.PA PNF?03.FT--CHAINS WITH PNSF3.PA PNF101.FT,PNF103.FT: LO PNF10?,PNLLSQ(HO)$*ADDPLT$ 1) UP TO 100 POINTS 2) STORES ABSORBANCE DATA ON RKBO (3A6 FORMAT): TIME-ABSORBANCE-STD DEV OF ABSORBANCE 3) PLOT DATA ON ADDS AND/OR CALCULATE SLOPES OVER SELECTED TIME INTERVALS PNF401.FT: LO PNF401,PNPR1(0)$*ADDPLT(L)$ PNF403.FT: LO PNF403,PNPR3(0)$fADDPLT(L)$ l) 100 POINTS ONLY 2) STORES ABSORBANCE DATA ON RKBO-AS ABOVE 3) PLOTS (ADDS) ABSORBANCE AND/OR FIRST DERIVATIVE OF ABSORBANCE VS TIME. USES SAVITZKY-GOLAY ll-POINT SMOOTH TO CALCULATE THE FIRST DERIVATIVE PNF801.FT,PNF803.FT: LO PNF80?,PNLLSQ(HOI)$*ADDPLT(L)$ 1) UP TO 100 POINTS 2) STORES ABSORBANCE DATA ON RKBO AS ABOVE 3) CALCULATES SLOPE OF ABSORBANCE FOR STANDARDS AND UNKNOWNS AND CALCULATES CONCENTRATIONS OF UNKNOWNS 4) PRINTS OUT RESULTS ON DECWRITER PNT?01.FT--CHAINS WITH PNSFT1.PA PNT?03.FT--CHAINS WITH PNTSF3.PA PNT101.FT: LO PNT101,PNTPL1(HOI)$*ADDPLT(L)$ PNT103.FT: LO PNT103,PNTPL3(HOI)$*ADDPLT(L)$ 1) UP To 100 POINTS 2) STORES ABSORBANCE AND TEMPERATURE DATA ON RKBO (5A6 FORMAT): TIME--ABS--STD DEV ABS--TEMP--STD DEV TEMP 3) AVERAGE OR NOT AVERAGE THE RUNS 4) PLOT (ADDS) ABSORBANCE AND/OR TEMPERATURE DATA 5) LIST DATA ON DECWRITER PNOD?.FT--OPERATE ON DATA FILES STORED (3A6 FORMAT) on RKBO INDEPENDENT VARIABLE=COLUMN 1 DEPENDENT VARIABLE=COLUMN 2 STD DEV OF DEPENDENT VARIABLE=COLUMN 3 THE PROGRAMS WERE WRITTEN ASSUMING TIME HAS THE INDEPENDENT VARIABLE AND ABSORBANCE WAS THE DEPENDENT VARIABLE. FILES WITH OTHER VARIABLES CAN BE OPERATED ON CORRECTLY BY THESE PROGRAMS, AS LONG AS THEY ARE VI. 133 IN 3E13.6 FORMAT. ONLY THE OUTPUT LISTINGS WILL BE LABELED INCORRECTLY. PNODl.FT: LO PNOD1(HOI)$*ADDPLT(L)$ 1) PRINTS OUT DATA ON DECWRITER AND/OR PLOTS DATA ON ADDS TERMINAL PNOD2.FT: Lo PNOD2(IO) l) COPIES DATA FILE FROM RKBO TO FLP2 BUT WITH FORMAT: 'RD',3F15.8 FOR EASE IN TRANSFER TO 11/40 COMPUTER VIA TTR811 PNOD3.FT: LO PNOD3,PNLLSQ(HOI) 1) CALCULATES RATES (SLOPES) FROM THE DATA AND PRINTS OUT THE RESULTS ON THE DECWRITER. PNOD4.FT: LO PNOD4,PNLLSQ(HOI) 1) DOES A REACTION RATE ANALYSIS OF UNKNOWNS BASED ON THE REACTION RATES OF KNOWNS (CONCENTRATIONS). 2) DATA FILES ON RKBO ARE DESIGNATED AS KNOWNS OR UNKNOWNS BY THE USER PNOD5.FT: LO PNOD5,AXIS,XYSYS(HOI) 1) PRINTS DATA ON DECWRITER AND/OR PLOTS DATA ON X-Y RECORDER PNOD6.FT: LO PNOD6(HOI) 1) CALCULATES SLOPES OVER SELECTED TIME INTERVALS USING AN 11-POINT SAVITZKY-GOLAY QUADRATIC SMOOTH. 2) PRINTS OUT THE SLOPE AT EACH DATA POINT PNOD7.FT: LO PNOD7(I)$*ADDPLT(L)$ 1) PLOTS ABSORBANCE AND/OR FIRST DERIVATIVE OF AB- SORBANCE (RATE) ON ADDS 2) USES SAVITZKY-GOLAY ll-POINT, QUADRATIC SMOOTH TECHNIQUE 3) CALCULATES AVERAGE OF THE RATE OVER SPECIFIED INTERVAL ' PNODT?.FT--OPERATE ON DATA FILES STORED (5E13.6 FORMAT) on RKBO INDEPENDENT VARIABLE=COLUMN l DEPENDENT VARIABLE=COLUMN2 STD DEV OF DEPENDENT VARIABLE=COLUMN 3 DEPENDENT VARIABLE=COLUMN 4 STD DEV OF DEPENDENT VARIABLE=COLUMN 5 134 THESE PROGRAMS ARE WRITTEN ASSUMING TIME IS THE IN- DEPENDENT VARIABLE AND ABSORBANCE AND TEMPERATURE ARE THE DEPENDENT VARIABLES. FILES WITH OTHER VARIABLES CAN BE OPERATED ON CORRECTLY BY THESE PROGRAMS AS LONG AS THEY ARE IN 5A6 FORMAT. ONLY THE OUTPUT LISTINGS WILL BE LABELED WRONG. PNODT1.FT: LO PNODT1,AXIS,XYSYS(HOI)$*ADDPLT$ 1) PLOTS TEMPERATURE AND/OR ABSORBANCE VS TIME ON THE ADDS AND/OR AN X-Y RECORDER 2) PRINTS DATA ON THE DECWRITER. APPENDIX C DIALOG FOR PAL8 PROGRAM WHICH OPERATES THE STOPPED-FLOW AND ACQUIRES DATA The printout halts at the appropriate times to accept responses from the experimentor. The experimentor's re- sponses are not shown in the dialog. DO VOU NISH TO USE THE PREVIOUS 180 ZT (BLHNK) END 9 ZT LEVELS? IF VOU 00. NONE SURE THE LIGHT SOURCE SHUTTER IS OPEN HND THE DRIVE SVRINGES HNE EHPTV HND DO NOT CHHNGE HNV OF THE PREVIOUS SETTINGS. TVPE '1' FOR VES OR TVPE '2' FOR NO: ---s:crro~ 1--- TVPE HNV CHHRHCTER HFTER VOU COMPLETE EHCH INSTRUCTION SET HND HRE REHDV FOR THE NEXT INSTRUCTION SET. UNLESS H SPECIFIC RESPONSE IS REQUIRED. (1) SET THE KEITHLEV OFFSET SNITCH TO 'LOC' HND THE STOPPED- -FLON TO HHNUHL. SET THE KEITHLEV HHPLIFICHTION SO THHT THE HHRLITUDE 0F CHHNGE (INCLUDING NOISE) FOR THE HOST INTENSE REHCTION IS JUST UNDER 9V. SET THE OFFSET RHNGE SO THHT iOO NT CHN BE OFFSET TO BELON +5V NITH NO HORE THHN 6.5 TURNS CLOCKNISE ON THE OFFSET 'FINE' KNOB. CONSIDER THE NOISE HND DRIFT HND NOTE ITS HHXINUH HNPLITUDE (IN VOLTS). CLOSE THE LIGHT SOURCE SHUTTER HND SET THE KEITHLEV OFFSET SNITCH TO 'REH'. (2) HHHT IS THE HHXINUH HHPLITUDE OF THE NOISE HND DRIFT IN TENTHS OF H VOLT? TVPE ONE OR THO DIGITS. THEN H SPHCE. NOISE= (3) OPEN THE LIGHT SOURCE SHUTTER. HHKE SURE THE DRIVE SVRINGES HRE EHPTV. THEN SET THE STOPPED-FLON TO HUTONHTIC. 135 136 ---secr10~ II--- (1)TIME INTERVHL BETNEEN HNHLOG POINTS: H=O.2. B=1. C=5. D=iO) E=106 MILLISECONDS (2)NUHBER OF HNHLOO POINTS HVERHGED FOR EHCH DHTH POINT: 3:1: 3:3: C=lO. 0:35: E=188 (3)TIME INTERVHL BETNEEN THE CENTERS OF DHTH POINTS IN UNITS OF THE TIME SPHN OF ONE DHTH POINT: H=1. O=10. C=iOO. D=1008 (4)NUMBER OF DHTH POINTS TO BE THKEN: H=10. B=SO. C=100) D=SOO. E=1608 TVPE THE PROPER LETTER HFTER THE HPPROPRIHTE NUMBER. (1): <2>= (3): <4): (5) HON MHNV TENTH’S OF H MILLISECOND BETNEEN THE TRIGGER HND THE STHRTING OF THE CLOCK FOR THE FIRST HNHLOG POINT? NOTE THHT IT NILL THKE ONE HNHLOG POINT TIME INTERVHL HFTER THE CLOCK STHRTS BEFORE THE FIRST POINT IS THKEN. (HIGHEST 4-DIGIT NUMBER=4095. HIGHEST S-DIGIT NUMBER=409SG) TVPE H NHOLE NUMBER THEN H SPHCE (IF LESS THHN S DIGITS): (6) NUMBER OF PUSHES PER SVRINGE FILLING= (7) RINSE STOPPED-FLON (THREE FLUSHINGS)? VES=1. NO=2 (8) BLHNK OR SHMPLE RUN: HLHNK=1. SHMPLE=2 (9) NUMBER OF SVRINGE FILLINGS PER RUN= (10) NUMBER OF DHTH PUSHES PER FILLING (MHX=5)= CHECK STOPPED-FLOR SVSTEM) THEN HIT HNV KEV. NHICH FORTRHN SHVE FILE DO VOU NISH TO CHHIN TO? IF THE FILE NHME IS LESS THHN 6 CHHRHCTERS. FILL IT OUT WITH “CIRCLE H' TO MHKE 6 CHHRHCTERS. TVPE H '?' TO STHRT THE NHME OVER IF VOU MHKE H MISTHKE. TVPE THE FILE NHME. THEN H PERIOD: APPENDIX D PAL8 PROGRAM, PNSF1.PA, WHICH OPERATES THE STOPPED-FLOW AND ACQUIRES SPECTROPHOTOMETRIC DATA The Program is shown in the form of a CREF* listing with the cross-reference table at the end. The first column is the CREF reference number and the second column is the computer core location. Core locations from 41008 and up contain 6-bit ASCII code for the dialog given in Appendix C and that section of the Program was not reproduced here. *See "OS/8 Handbook", Digital Equipment Corporation, Maynard, MA. 137 138 .000?0 0000 00hz020¢0znlmzmhzb00\ .00092000 z— mHDA<> 02—Hm<400.¢000—flblbm0m 00h HP<¢ MO0J0\ 0000 .0000eflh flflbh< 092000 30040 00 H sz~£\ 000k .mm>4¢> .h.m 000 MHZD00\ 000k 20000 00 u sz—=\ Grub .0z—Ad_h 000 000000 00 R 002—2\ 0000 .QOZ—Hmbdh 00Z~fl>m 00 t sz—E\ vbbh .>¢~P~m0m 0E ZO—mzm>200\ 000* .Abbbbudm>md PAD<0000 40>04 Mz<00\ bhbh 000NHMZ¢AQ .0flmNz0ZumAm=m 00 t\ 0 .—I 0040090 b= h0vbu~>fi .0< £00 dflhmn0fl¢ 040020 M0000 fldmd0\ 00—0HHNA0 .PmDfiflHFZ— 20000 :0 0~Mm\ ~0—OHM040 .oidh.¢<040 0 00 0F mDPP—mmm>.zb mFde z<0nm0ufl I N80: 920M m—AA—mm\ 0m.~hmzh «2000000 BOJEIGHHQOFQ — H040 0h\00\00 no>lwd0 0000 0H9ZH=000zulmmm9zD00\ .00092000 I— 0004¢> 0zu9m§9m\ .mmmwbm <9<0 00 u mDZ~S\ .0040: 0000H09I9m00 000 0900 M0040\ .0000—09‘00900 09:000 M0040 00 \ sz—=\ 000000000000 00b- 00b- 00b- blah- .mm>4¢> .h.mA¢0h $92000\ 00hb M0040 00 \ 002—2\ 0000 .00—44mm 000 000000 R0 \ mDZ—E\ 0bhh .m0z—0004m 002—090 00 & mDZ—=\ 0hbh .>MdZ—0 H>~9mm0m 09 z0-mmm>z00\ 0000 .Ahbbbu40>04 94D04 Mz<40\ bhhk .0mflNuMz<40 .000NZOZn04m=¢m\ 0 .0000 000 sz—0m <90: L0 \\ 00— .09Z~00 0900 3003900 09—20 02—9\ 0— .90—00 <9a0 000 092—00 004m 00 §\ 0 .gl 0000090 <9<0 00h z0-9d004 0!—9¢<9Q\ 000 .0000090 ¢9<0_¢0h z0~9<004 9000000\ 0 b—00fi h00hu<030 ~00hu<0m ——0hu~=z 0—0hu4flm h—0blmm4 000hu>b= 900hnm>0 .0< 000 0090—000 040020 M0040 04040\ 00—0n0040 .900000920 M0040 B0 m—Mm\ ~0~0uMm40 .0040 04040 0 0< 09 009090 M0040\ 00—0u0040 .0< 000.009mu000 040¢z0 M0040 9mm\ 00—0u0040 .HHmfimmmehhbm M0040 09 0¢\ 00—0u0<40 .000! >9~m¢m>~z0 09090 H¢0~00~2 I N90: 920M m—44—00\ ¢Q._hmzm »=<00000 304b|0wmm09m — 0000 0b\00\o0 00>I04dm QA m90~>mflm mmm¥\ .d0901h 50 fizmzomxm\ .m—OFU<.m Pmmhh0\ .~ BO—Pomm flmhhd Emma mHEmDm mDZ—=\ .P40> < ho mmhzmblqufl¢E\ .mFJO>Iz—Qm<=\ .AH>MA FR 0\ .4thO an z_um¢z mmmmb\ .0¢03 F—mI¢N\ HDA<> 23m Mz<4n\ .mm>4<> .b.m ¢0h\ mPZDOD MUOAU\ .wrz—om fl<fl0mflflh\ .nm03 P—fllVN\ 090<\ .mmoqblmnqh 0>A¢> 0<000\ .000N 0>00< 00000 00000 030 0000 0:000 030 ~00: 0£P00 000: 00I0 0+. ~00: 000: ~00: 0+. 000I0 000: ~00: 00I0 000: 0000 0~0<0 0+. 000I0 ~00: 0000 0~00 0000 0~00 M00 ~ 000 0000 0~I0 <00 000 ~000 open .nouc .nwo ea.» been e.su hoe. .auu .ueo ne¢n ~uou «no. noon .uup «no: new» _~¢u _uo_ been «new .ua. s_aa _uem «new .mo. wean ov+~ an”. «no. .nea .um. .man .auuv mum. enau s¢vu mum. .uo. unoo can. puoe .223 000~ ~000 0000 0000 0000 .0000 000~ 00000 ~0000 00000 00000 00000 00000 00000 00000 00000 ~0000 00000 0~000 0~000 0~000 0~000 0~000 0~000 -000 0~000 00000 00000 00000 00000 00000 00000 ~0000. 00000 00000 00000 00000 00000 00000 00000 ~0000 00000 00000 00000 00000 00000 00000 00000 ~0000 00000 00000 00000 00000 00000 ~0~ 00~ 00~ 00~ 00~ 00~ 00~ 00~ 00~ 00~ ~0~ 00~ 00~ 00~ 00~ 00~ 00~ 00~ 00~ 00~ ~0~ 00~ 00~ 00~ 00~ 00~ 00~ 00~ 00~ 00~ ~0~ 00~ 00~ 00~ 00~ 00~ 00~ 00~ 00~ 00~ ~0~ 00~ 00~ 00~ 00~ 00~ 00~ 143 .00000 0090\ .N0 00~ 9< 000<9\ 80—00 0I< 00~\ 00 H0<0H>< 00~h\ 0NAO <00 <40 0A <00 A>A <00 000 <00 9000 0000 0000 0900 00>0 0m>0 m<00 0000 A000 A>AON 0>AOJ 0~000 000 000 <00 0<9 <00 0<9 0:0 <00 0<9 <00 <00 009 <00 0<9 020 009 .0>A0< 00<0 00~0 0000 00~0 000~ 0000 000~ 0000 ~000 0000 0000 .0000 0000 0000 0000 ~000 0000 ~0~0 0000 0000 0~00 0~00 0000 0000 0000 0000 ~0- 00- 0000 00- 000~ 00- 00- 0000 .0000 0000 .000~ 0000 .000~ .0000 .0000 .000~ .0000 .~000 000~ ~000 .000~ .0000 000~ -000 0~000 00000 00000 00000 00000 00000 00000 ~0000 00000 00000 00000 00000 00000 00000 00000 ~0000 00000 00000 00000 00000 00000 00000 00000 ~0000 00000 00000 00000 00000 00000 00000 00000 ~0000 00000 00000 00000 00000 00000 00000 00000 ~0000 00000 00000 00000 00000 00000 00000 000 000 000 000 000 000 000 000 ~00 000 0~0 0~0 0~0 0~0 0~0 0~0 0~0 0~0 -0 0~0 000 000 000 000 000 000 000 000 ~00 000 00~ 00~ 00~ 00~ 00~ 00~ 00~ 00~ ~0~ 00~ 00~ 00~ 00~ 00~ 00~ 00~ 00~ 00~ 144 m< EFF? M04n< 4 mtfi mtm <40 .fl0<fifl>< NIP In MPH—Om h\ v?— ~>= 4>4 G4 mlfi 4n<0 NW— 440 4N0 4>4 <0é 4>4 n4 .4>4 Nana floa— oun— mama Oman aneh Ouch N—Ob ON—b Nana 4990 0900 @600 6600 @666 each uflhh vv—O hcvh unfl— ~N¢b 4nN— OQNN ou—o Ova O—ND mnNN 66—h NmNN vab unflm gna— 66—h ~00— vanv wNNb Nano _@n@ ocmh Dane 04“” 4040 ”940 N040 0N0— Ocmb bov90 covoo novco fiovoa movoo NoveO novOO oo¢oo unvao on¢®0 unveo vmfioo mm¢00 Nnvoe unwoo onvco N¢VOO o¢¢os n¢¢00 ¢vvee mvfios NGvOO «eves QV¢OO hmvso omvoo mm¢oc vmvoo moveo waves umveo emvcc NN¢®O eaves mN¢oc vN¢Go mw¢eo Nflfiao ~N¢Qo ®N¢®Q huvoo 04060 D—woo v—QQO m—vos N—¢QO nbN 0h“ aha NNN «hm CNN 69N 00N how @ON new vefl ”ON NON ~0~ sow oafl @nN hnfl on“ now $9“ an“ «an 4n“ cum 6*“ 0¢N hvfl 0+N ova va avN Nvfl nvfl O¢N on“ 00~ haw owfl nmN tmN man N0“ 40“ ea“ 145 ENG 500 Pfimhho m~\ .Pfimhh0 mm4n< w mam mu =44\ .Qfll mu 4>4n< 0 mow m— :44\ .mém MPOQ m0 002\ 0900 Hfl< 4>4n< 0 E44\ .002 m— :44\ .mon 04 =44\ .0< mum 404 Q404 <06 4>4n< mlfi unvo m4o4 Q404 Q404 <00 4>4Q< mZH 4n¢o m<94 Q499 9499 <09 0000 0N000 000 4>49< mlfi .hhbv h~000 900 ~0v0 ~000 0~000 000 m<99 9404 0000 90000 0N0 0 .0~94 0000 00000 bN0 0 .m<94 0000 N0000 0N0 0 .244 0000 ~0000 0N0 B994 920 0000 00000 0N0 m<94 <09 N000 90000 0N0 m<94 9

499 0~99 090 N000 090 ~000 .9949 .9999 .90~9 .0090 N000 .9090 ~000 0009 00~0 .0990 090~ .~990 N90— 0000 0000 0000 0000 .0990 .0990 090~ 0009 0000 0009 00N0 0009 00N0 00N9 99N0 0009 090~ 000~ 0009 09N0 0~09 000~ 000~ 0000 .9990 ~000 000~ 00N0 000~ 0009 00N0 00N0 0009 000~ 00N9 0N900 9~900 0~900 0~900 0~900 0~900 N~900 -900 0~900 90900 00900 00900 00900 00900 N0900 ~0900 00900 99000 09000 09000 09000 09000 N9000 ~9000 09000 90000 00000 00000 00000 00000 N0000 ~0000 00000 90000 00000 00000 00000 00000 N0000 ~0000 00000 90000 00000 00000 00000 00000 N~0 -0 0~0 000 900 000 000 N00 ~00 000 000 900 000 000 000 000 N00 ~00 000 000 000 900 000 000 000 ~00 090 090 990 090 090 090 090 N90 ~90 090 000 000 900 148 .099 9<940\ .099 990\ .N99 9<940\ .~99 9<940\ 4>49< 090 4990 990 9~499 090 <90 09900 919 ~9F 090 0|. 990 0900 ~9F 090 0000 N900 ~98 090 ~900 .4990 90<9 00N0 ~000 .099~ 0009 .0990 0009 090~ .099~ .0990 .099~ ~009 0009 0NN~ ~009 .099~ .0990 .9990 .000~ 0000 ~000 0NNO 00N0 0999 NNO~ 0N~0 00NN 000N 0N0~ 000~ .0090 00N0 .009~ .0090 ~009 900~ .9090 0N00 .~99N 0900 .9090 0000 .9090 N900 .9090 ~900 ~N0—0 0N0~0 9~0~0 0~0~0 0~0~0 0~0~0 0~0~0 N~0~0 -0~0 0~0~0 900~0 000~0 000~0 000~0 000—0 N00~0 ~00~0 000~0 99900 09900 09900 09900 09900 N9900 ~9900 09900 90900 00900 00900 00900 00900 90900 00900 00900 00900 00900 N0900 ~0900 00900 9N900 0N900 0N900 0N900 0N900 NN900 ~N900 000 000 N00 ~00 0N0 0N0 9N0 0N0 0N0 0N0 0N0 NNO ~N0 0N0 0~0 0~0 9~0 0~0 0~0 0~0 0~0 149 .9998 0<9 9099 E0<90 90 \u~999\ #09994 9.994 99. 90>9 9.9~90\. 99>9 90>9 90>9 ~999 ~999 90>9 99F ~>99 99h 90>9 40: 9999909990 99~>~9\ 09590 9949 49499 0 0049 ~ 0~ 49499 0 0049 ~ 0~ 4>499 0 0049 ~ 0~ 9999 0 0049 9 0~ 9P09 0 0049 ~ 0— 0<99 0049 99~00 440 0000 0N00 0000 0~09 0N0~ 000N 0000 ~009 0N00 -09 000~ ~000 0009 000~ 0N0~ 00~9 ~N09 9009 .9999 0000 .909 .9949 .9990 .~990 0000 .099~ ~0N0 9~00 -N0 090~ ~0N0 9~00 -N0 .099~ ~0N0 9~00 -N0 090~ ~0N0 9~00 -N0 090~ ~0N0 9~00 -N0 .099~ 9~00 N90~ 0009 0000 0000 00-0 990~0 090~0 090~0 090~0 090~0 N90~0 ~90~0 090~0 900~0 000~0 000~0 000~0 000~0 N00~0 ~00~0 000~0 900~0 000~0 000~0 000~0 000~0 N00~0 ~00~0 000~0 900~0 000~0 000~0 000~0 000~0 N00~0 ~00~0 000~0 900~0 000~0 000~0 000~0 000~0 N00~0 ~00~0 000~0 9N0~0 0N0~0 0N0~0 0N0~0 0N0~0 NNO~0 150 .alf9<940 999B 909H9099 0< 9<04\ 30499990 99~9~9\ .9990: 099 999 090<90 90 huN999\ .dfln99 49~P0 4999 b99\ ..~.~9~4 9.994\ 9<9b 0094 990 09 999 99~90\ .0<_9<fl40 999b..oé.9099 a: 9<04\ 2&099 .9999 .~999 0000 0090 0090 ~000 9900 99~0 00N~ 0000 0000 0000 000~ ~009 000~ N000 ~N99 N009 0009 9009 9~09 000— 0000 00N9 0N00 000~ 0000 ~009 0000 -09 000~ 0N00 0009 000~ 000~ ~N09 ~00~ 0009 99-0 09-0 09-0 09-0 09-0 N9-0 ~9-0 00-0 00-0 00-0 00-0 N0-0 ~0-0 00-0 90-0 00n~0 00-0 00-0 00-0 N0-0 ~0-0 00-0 9N-0 0N-0 0N-0 0N-0 0N-0 NN-0 ~N-0 0N-0 9-~0 0-~0 0-~0 0-~0 0-~0 N-~0 --0 0-~0 90-0 00-0 00-0 00-0 00-0 N0-0 ~0-0 ~00 000 000 000 900 000 000 000 N00 ~00 000 000 000 900 000 000 000 N00 ~00 000 0N0 0N0 9N0 0N0 0N0 0N0 0N0 NNO ~N0 0N0 0~0 0~0 9~0 0~0 0~0 0~0 0~0 N~0 -0 0~0 000 000 900 151 .994.909 990F0\ 99909 89 0~\ 99 9< P~ 0~\ 99 < P— 0~\ 90 < 99 0~\ 99 < b— 0~\ 9< 9< P— 09\ .994.909 99090\ .999.9099 0909990 9999—\ 9009 00<0 II. 8' 8' V U _ 8 ' U ~I0 fl ~00I0 N90 N90 9094 9009 ~9903 ~9 ~l0 ~I0 ~0 ~I0 ~9 ~lv ~< ~00Iv ~90 ~90 90~4 9009 ~0<0 .N9P90 .0990 90<9 .0990 N00~ .0090 .0090 0009 090~ .0090 0009 090~ .0090 0009 090~ .9090 0009 090~ .0990 0009 090~ 0-~ 0-0 00~0 .0990 ~90~ .N990 .0990 0009 090~ 0000 0009 090~ 0000 0009 090~ 0N00 0009 090~ NN00 0009 090~ 0-~ 0-0 .~9F90 00~0 .0990 990~ .09F90 0009 00N~ 00N—0 00N~0 NON~0 ~0N~0 00N~0 90N~0 00N~0 00N~0 00N~0 00N~0 NON~0 ~0N~0 00N~0 90N~0 00N~0 00N~0 00N~0 00N~0 NON~0 ~0N~0 00N~0 9NN~0 0NN~0 0NN~0 0NN~0 0NN~0 NNN~0 ~NN~0 0NN~0 9~N~0 0~N~0 0~N~0 0~N~0 0~N~0 N~N~0 -N~0 0~N~0 90N~0 00N~0 00N~0 00N~0 00N~0 NON~0 ~0N~0 00N~0 900 000 000 000 000 N00 ~00 000 000 000 900 000 000 000 000 N00 ~00 000 090 090 990 090 090 090 090 N90 ~90 090 000 000 900 000 000 000 000 N00 ~00 000 000 000 900 000 000 000 000 N00 152 N~0 ~l0 ~I0 0< ~00I0 090 090 90~4 .9009 00<0 .~0 .~9 .~< .09090 .0990 0NNO N000 000~ 0NNO NN00 000~ 0N00 NN00 000~ .0090 .9090 0009 090~ .0090 0009 090~ .~090 0009 090~ .N090 0009 090~ .0090 0009 090~ 0N- 0N~0 00~0 .0990 000~ .0090 .0090 0009 090~ .9090 0009 090~ .0090 0009 090~ .~090 0009 090~ 9-~ 9-0 .09990 00~0 N00~0 ~00~0 000~0 9N0~0 0N0~0 0N0~0 0N0~0 0N0~0 NNO~0 ~N0~0 0N0~0 9~0~0 0~0~0 0~0~0 0~0~0 0~0~0 N~0~0 -0~0 0~0~0 900~0 000~0 000~0 000~0 000~0 N00~0 ~00~0 000~0 99N~0 09N~0 09N~0 09N~0 09N~0 N9N~0 ~9N~0 09N~0 90N~0 00N~0 00N~0 00N~0 00N~0 NON~0 ~0N~0 00N~0 .90N~0 00N~0 00N~0 000 N00 ~00 000 000 000 900 000 000 000 000 N00 ~00 000 0N0 0N0 9N0 0N0 0N0 0N0 0N0 NNO ~N0 0N0 0~0 0~0 9~0 0~0 0~0 0~0 0~0 N~0 -0 0~0 000 000 900 000 000 000 000 N00 ~00 000 600 900 153 00 999 ~0 999 ~9990 990 9009 090 0~0<0 9<9 .99999 09093\ 440 <40 N990 990 9040 <09 009~ v 9<9 N990 990 9040 <09 00~0 9<9 .N< .9990! .~fl 90<9 .~990 0N00 090~ .~990 0N00 N90~ .0990 .0990 090~ 0009 .0990 990~ 000~ 00~0 0000 9909 9999 000~ 000~ N900 900~ N~0~ 0~0~ 0N0~ 0N0~ 0N0~ 9900 000~ 000~ 000~ 000~ 000~ 0000 N00~ 000~ 000~ 000~ 000~ ~90— N000 N~00 N000 00~0 0NNO NN00 N00~ 0~0~0 0~0~0 N~0~0 -0~0 0~0-0 900~0 000~0 000~0 000~0 000~0 N00~0 ~00~0 000~0 990~0 090~0 090~0 090~0 090~0 N90~0 ~90~0 090~0 900~0 000~0 000~0 000~0 000~0 N00~0 ~00~0 000~0 900~0 000~0 000~0 000~0 000~0 N00~0 ~00~0 000~0 900~0 000~0 000~0 000~0 000~0 N00~0 000~0 000~0 000~0 000 000 900 000 000 N00 ~00 000 090 090 990 090 090 090 090 N90 ~90 090 000 000 900 000 000 000 000 N00 ~00 000 000 000 900 000 000 000 000 N00 ~00 000 000 000 900 000 000 000 154 .90000 00003\ .90000 00003\ .90000 05003\ fl900 000m 0am<0 400 00: 009-0 09m 00: 0090 090 m0: vv—u 90: N00 090 00: N~0 0:900 0000 0—mdv 440 3900 0—9 0D9—0 9900 0—9 $0—0 9900 009 N—V 0900 009 qw "£900 000m 0—m<0 400 0900 001 90—0 0900 00¢ 000 0900 001 N00 .0900; .00 .10 .10 .v0 .04 .90 .av .ad .9900; .NH .N0 .9090 .0990 D90— 0009 09Na 990- a9fln 0&00 000— e9fla 0N00 000~ a9fln 0N00 ~00— a9flb 0N00 900— .N090 .099? 690. 0009 .0090 ”N00 990— .0090 DN00 now— .0090 0600 900— .ao9n N90— .0090 .099! D9”— 0009 .~990 0~00 000— .099” 0N00 000~ .~990 0N00 900— 090~0 090~0 N90—0 —9v—0 090—0 900~0 000~0 ”00~0 000~0 900~0 NOQ—O ~00—0 000~0 900~0 00000 nDO~0 Gav—0 000~0 Nbv-0 ~00—0 000~0 900~0 009—0 DOG—0 090~0 090~0 «Qt—0 009—0 0¢vu0 900—0 oat—0 ”av—0 900~0 000~0 N00~0 ~0v—0 000~0 9N0~0 060—0 an—0 Gav—0 ”NO—0 NNO—0 ~N0-0 0N0~0 9—v—0 0~0~0 a.v.o, 009 909 009 009 #09 009 N09 ~09 009 039 099 9N9 0N9 069 $09 NN9 —N9 0—9 0—9 9—9 009 n—9 v—9 0—9 N—9 -—9 0~9 009 009 909 009 009 $09 009 N09 n09 600 000 900 090 000 $00 000 N60 ~00 155 .0H50 90S9ml0l\ 9000N d 9~ m~\ .0000lmfl9\ 9fludmm 4 9~ m~\ .0_¢0 H0100 H0 9mfili0<00 09h~h\ .9~0~0 090000 900\ .9~0~0 00~09 900\ .9~0~0 000000 900\ .9~0~0 9mfl~h 900\ §§E§§Eé§ $2§§é駧§§§ 0:0 049 0:0 <00 009 000 430 0§9 mlfi 0<9 mlfi mlfi 049 <00 mlfl <00 mflfi 0§9 <00 <00 <00 <00 <00 <00 <00 <00 <00 <00 mlfi 009 .~099 .090 9~9~ 9N9~ 0~0~ 0009 ~00~ 0099 000~ 00~0 9980 .0990 000~ 0009 .0090 0009 000~ .9090 0009 000~ 00~0 .~090 0N- 0N~0 00~0 .~090 0N- 0N~0 00~0 .~090 NN- «N~0 00~0 .~090 ~N- ~N~0 00~0 .N090 .0090 .0090 .0090 0N~0 0N~0 ”N~0 «N~0 ~N~0 .0990 000~ 000~0 N00~0 ~00~0 000~0 900~0 .ova_o 000~0 000~0 N00~0 ~00~0 000~0 900~0 000~0 000~0 000~0 000~0 «00~0 ~00~0 000~0 9N0~0 000~0 0N0~0 0N0~0 0N0~0 NNO~0 ~fl0~0 0N0~0 9~0~0 0~0~0 0~0~0 0~0~0 0~0~0 N~0~0 -0~0 0~0~0 900~0 000~0 000~0 000~0 000~0 N00~0 ~00~0 000~0 990~0 090~0 090~0 009 009 «09 ~09 009 099 099 099 099 099 N99 ~99 099 009 009 909 009 009 009 009 N09 ~09 009 009 009 909 009 009 009 009 N09 ~09 009 009 009 909 009 009 009 009 N09 ~09 009 009 156 .9~0~0 000009 4 900\ .539: 5.5:: 8. bags? finance? 33.6 < .5 9x 0033 ~ 09~0 440 0400 N~l0 ~090 0000 0~m40 000M0 0010 0~00h 00NI0 0000 00000 0000 00000 .0~0>b .0~00h 0040 0~00 0090 0009 9~00 0009 090~ .~990 .0990 090~ 0009 0000 0009 090~ 0000 0009 090~ 0000 ~0N0 9000 090~ 00~9 ~0N0 9000 990~ 00~9 000~ 009~ 000~ ~0~0 000~ ~000 00N~ 0~00 00~0 00N~ 090~ 000~ 0000 0090 99N~ -00 009~ 909— 000~0 900~0 000~0 000~0 000~0 000~0 NNO~0 ~00~0 000~0 9~0~0 0~0~0 0~0~0 0~0~0 0~0~0 N~0~0 -0~0 0~0~0 900~0 000~0 000~0 000~0 000~0 N00~0 ~00~0 000~0 990~0 090~0 090~0 090~0 090~0 090~0 ~90—0 090~0 900~0 000~0 000~0 000~0 000~0 000~0 ~00~0 000~0 900~0 000~0 000~0 000~0 157 .9EJHO 0H00~09J9000 H94400040\ .m9~0~0 0 00 9% 909~0—0 0 9~ 0~\ 909~0~0 039 9~ 0~\ 99~0~0 000 9~ m~\ 994400 0000~09I9000\ 0~ 09~0—0 :4: Ba\ 009~0 .009~0 .009~0 .809~0 .~09~0 .0000 0009 ~N09 000~ NN- 0000 ~N99 0009 ~N09 000~ ~0~— 9000 000~ 9~00 000~ 9000 000~ 0000 000~ 0900 9~00 000~ 9000 000~ 0000 000~ 0900 9000 000~ 0000 000~ 0900 0000 000~ 0000 0009 0000 0009 900~ 0000 0009 900~ 0000 0009 900~ 0N- 909~0 009~0 009~0 009~0 009~0 009~0 ~09~0 009~0 990~0 090~0 090~0 090~0 090~0 090~0 ~90~0 090~0 900~0 000~0 000~0 000~0 000~0 N00~0 ~00~0 000~0 900~0 000~0 000~0 000~0 000~0 N00~0 ~00~0 000~0 900~0 000~0 000~0 000~0 000~0 N00~0 ~00~0 000~0 900~0 000~0 000~0 000~0 000~0 N00~0 ~00~0 158 040K 40M0 000I0 000 000 00.4 900~00~h 000~090_000\ 0000 000000 90 h 9H0\ 0040 09b: 009: 030 00NI0 0092 009! 030 00Nl0 009: 0092 030 400 049 049 400 049 440 0040 .0000 0000 0000 0009 0099 ~0~0 0000 990~ 0099 9999 ~000 0~00 00~0 009~ 0009 9990 0000 0000 .9090 .0090 0009 000~ 00- 0N~0 .9000 00~0 .0990 ~00~ 0000 ~009 000~ ~099 .0Nl 0000 0009 ~N09 000~ 0N- 0000 000~ ~099 .0x0 0000 0009 ~N09 000~ 0N- 0000 000~ ~N99 999~0 099~0 099~0 099~0 099~0 N99~0 ~99~0 099~0 909~0 009~0 009~0 009~0 009~0 009~0 ~09~0 009~0 909~0 009~0 009~0 009~0 ~09~0 009~0 909~0 009~0 009~0 009~0 009~0 009~0 ~09~0 009~0 909~0 009~0 009~0 009~0 009~0 009~0 ~09~0 009~0 9~9~0 0~9~0 0~9~0 0~9~0 009~0 0~9~0 -9~0 0~9~0 000 000 000 ~00 000 0~0 0~0 9~0 0~0 0~0 0~0 0~0 N~0 -o 0~0 000 000 900 000 000 000 000 N00 ~00 000 000 060 990 090 000 000 159 9000 040040.00HMO¢40\ 90 4 90 00\ 90 d F— m—\ 9900 00 00000\ 0000 000 00<0 0!? 0000 <00 £00 0000 006 #900 0:5 005 $000 0000 <00 00000 009 000 000 0000 000 00000 009 000 400 00000 000 <00 0|. 0

A<> HBE>~P0¢\ —0! <0: ~0ll fish n¢00 400 <00 0 flmhm nth flmmmh mlfi Hum: N0— uth mlfi 9~00 uflfi mlfi 0000 —:F mun N500 uflfi nth ~h00 NIP mlfi N000 ml? mlfi ~000 0mm: 40a final! at? 00 min N+. min 0mm! NW— 440 £40 mall <05 an: as? man: <00 nah :45 Adz! <0: 0&1 ash had: 009 had n0 00—>~0 0z<\ PI—0m <5d0 500I0402 HIP 500 0P\ mfizm00 004400 hO Pflm\ HIP h0 fl0<¢0>< HIP HMSP\ .PB—Om QFEG fifih\ MPH—0.— 0004: 0000A\ 000240 b.l<0 «bim><0\ .00—bd002.>¢<¢00lflh\ In $200 001:: .500000\ .hfldfi—fl H>~b~000 OF_&0H>200\ .mrz—00 004500 0t<\ .ciAhAM0040 01000\ 00—0 0005 5000 n000 —000 5—00 -00 500— —005 0000 0005 0000 5005 500— 0000 000— 0000 0005 500— 0000 0000 5000 —055 0000 5005 000— ~005 000— 5005 0000 5000 00—5 0000 0005 0000 000. 00—5 ~00— 0000 0000 0000 ~000 0005 00~0 0000 .~0Jh ~0~0 0~000 0—000 0—000 0~000 —_000 0~000 50000 00000 00000 00000 00000 00000 ~0000 00000 55000 05000 05000 05000 05000 05000 ~5000 05000 50000 00000 00000 00000 00000 00000 —0000 00000 50000 00000 00000 00000 00000 00000 —0000 00000 50000 00000 00000. 00000 00000 00000 «0000 00000 000~ 000— 000- 000— 000— —00- 000- 000— 000- 500- 000— 000— 000- 000— 000— n00- 000— 000— 000— 500— 000- 000— 000— 000— 000- —00— 000— 0~0— 0~0- 5.0— 0~0— 0~0— 0—0— 0—0- 0—0— ——0— 0~0~ 000— 000— 500— 000— 000— 000— 000— ~00— 166 .4800 000010 FDLPDO 000\ 0000 040040.00U009000 0000 000PU€00\ .0>< Q4<0\ .0 04000 0 .00004 000' 00.0 0000 500— 000' 000— 0000 000— .0550 #50— 00~0 050— 0000 050— ~000 0005 .5550 0000 0055 0000 50v5 000— 0005 000— 0000 000— 0000 0000 0005 0000 0005 0000 000— 4000 500— 0000 0005 5400 000— 0000 0000 0000 ~00— ~000 0905 000— 05000 50000 00000 00000 90000 00000 00000 00000 00000 50000 00000 00000 #0000 00000 00000 00000 00000 5v000 00000 09000 ##000 0'000 00000 ~¢000 09000 50000 00000 ”0000 00000 00000 00000 00000 00000 50000 00000 00000 #0000 00000 00000 —0000 00000 50000 0~000 0~000 0~000 0~000 000— 000- 500- 000— 000- $00— 000— 000- 000— 000— 000— 000— 500— 000— 000— 900- 000- 000— 000— 000— 000— 0~0- 500— 0~0— 000— $40— 000~ 000— 000— 0~0— 000— 000— 500— 000— 000- 0000 000— 000— 000— 000~ 000— 000— 500— 000— 000— 000— 168 OBOE — mlfi whoa huh N+. LIB QB—Q NO— flmbh ml" 3&9? — &:6 war? mlfi O'Nv ash mm>h man fiuflv as? Hark mlh O mock mlh 39°F mlfi O—mo fish Ina? mlfi 0&0 as? :59? mlfi 0&0 Q8 mlfi OVNV as? flm>9 min ¢aflv ask an»? mlfi OONV fig? omca mlfi ud PR 00. and 0 H>flflh HmD\ .A>A>O .hmhm H040 0N00 —0NO huf— ——N0 .0bb0 ~0N0 N~0- ——N0 0500 «060 hav- nuflo §h00 ~0N0 kn!— ——N0 .ubha «060 h—0— —_NO h—00 «h0— n0fla .Ohhv 050— 0000 0u§b Gha— ~NND 0n$b aha- 00~0 .Oth kha— ~N00 0N00 0000 0N0fl ~000 0000 0080 00000 00000 #0000 0v000 «9000 uv000 00000 h0000 00000 60000 #0000 00000 «0000 ~0000 00000 NN000 0N000 ”N000 9N000 ”N000 NN000 ~N000 0N000 h—000 0—000 n—000 0.000 0~000 «~000 -000 0~000 50000 00000 00000 #0000 00000 «0000 ~0000 00000 hhbfl0 0~N~0 ebbfl0 Obhfl0 0bhfl0 000— 0N9— 0N0— NNO— 0N0— nut— 0N0— 00¢— NNO— ~N0— 0Nv— 0w?— 0~0— h—G— 0~0— 0~9— 0~0— 0n?— Nav- u—O— 0~0— 00v— 00*— D09— 00*— 000— 000— «00— ~00— 000— 000— 000— how— 000— 000— @00— 000— N00— ~00— 000— 000~ 000— baa— 000— 170 .flK—hbaflflbmlLfimrh 0 -OQ< 0? F>IU\ .Egm Emma—0 POE—.5: I‘m! 000 0000 8000. 2000 “mm 0000 N000. lflml nmm 0000 80000 N005 000- ~000 -00— N000 N00h 000— 0000 _00— 0000 0—0h “—08 000- 5000 ~00- 0000 000— 0000 IIHLP 0000 .0000 .00b0 bh0¢ #00— hh0f #00— 500* 000— 0000 000— 000v 0N0~ 000$ .0hhu 0000 000— 000— 000* .nhhu .0bbv 000— .N090 0N00 000N 0000 ~0N0 b—fn ——00 00—00 '0—00 00—00 «0~00 —N—00 00—00 bu—00 0-00 0——00 v~—00 0—u00 N-00 ———00 0-00 h0—00 00.00 00—00 00—00 00—00 N0—00 _0—00 00—00 hb000 0h000 0b000 vh000 0h000 Nb000 uh000 0b000 N0000 00000 00000 00000 00000 «0000 ~0000 00000 h0000 00000 00000 #0000 00000 «0000 ~0000 00000 b0000 NNO- 0&0— 050— 0b!— .0h0— Nb!— nbvu 0&0— 00¢— 00¢— N00— 00v— 00$— 00'— 000~ N00— ~00- 000— 000— 000— N00— 000— 00*— V00— 00'— N00— ~09— 00v— 000— 009— hQOu 090— 00¢— $00— 000— N00— 099— 009— 009— 00'— N00— 00¢— 00v— $09— 000— N00— ~00— 171 019 000 as? ESE .bhflo H040 .000 .000 ..00 .ZQMI h0fl00 00800 00000 #0000 00000 00000 .0000 bh.00 05.00 0b.00 0b.00 0b.00 05.00 .h.00 0b.00 h0.00 00.00 00.00 00.00 00.00 00.00 .0.00 00.00 h0.00 .0.00 00.00 hv.00 0v.00 00.00 00.00 00.00 «0.00 .§.00 00.00 h0.00 00.00 00.00 00.00 00.00 «0.00 .0.00 00.00 hfl.00 00.00 000. N~0. .00. 000. 0.0. 0.0. h.0. 0.0. 0.0. 0.0. 0.0. 0.0. ..0. 0.0. 000. 000. b00. 000. 000. $00. 000. N00. .00. 000. 00*. 00'. NOV. 00'. 00¢. 000. 000. Nov. .00. 000. 00'. 00v. 800. 000. 000. 900. 00$. 00'. .00. 00'. Obv. 0&0. 172 0000. . .Idflh005.09.l.<00\ .0 001% 000i. 000 . JAG hhm 0000 0.00. >0.hum H=<004.h 0000. . mlfi 0. 0.0 0 000 .ufld 000 M00 as? 440 PH! 02¢: .00: .0000 .0040 ..Qfi< 0000 0000 000. 000. 0000 000. 0000 0000 000. 0000 0000 .000 0000 000. 0000 000. 0000 00.0 0000 0000 .000 0000 0000 0000 0000 .000 .0.0 0000 .000 0000 0000 0000 0000 0.00 0000 0.00 .000 0000 000. 0000 0000 .000 00000 00000 .0000 0.000 0.000 0.000 0.000 0.000 0.000 ..000 0.000 00000 00000 00000 00000 00000 00000 .0000 00000 00000 00000 00000 00000 00000 .0000 00000 00000 00000 00000 00000 00000 00000 .0000 00000 00000 00000 00000 00000 00000 00000 .0000 00000 00000 00000 0.0. 0.0. 0.0. 0.0. 0.0. ..0. 0.0. 000. 000. 000. 000. 000. 000. 000. 000. .00. 000. 000. 000. 000. 000. 000. 000. 000. 000. .00. 000. 000. 000. 000. 000. 000. 000. 000. 000. .00. 000. 000. 000. 000. 000. 000. 000. 000. 000. .00. 000. 174 .00“ DDAL .O§ HIP Ivan hbm\ QNN as? war? mlfi NNN as? Mm: mlfi ¢n<0 — fish HUGO LES dado Nm— MMEA min NIL NM— flmhb mlfi ONN QSP bane mlfi Hmhh mlw ammo GS? 4A0 <40 ounvc a-¢0 O—«VO aunvo N—nve ———v0 O—qvo bO—fib cenio aO—vo GQ—VO mO—vo NO—OO ~0—v0 00—00 buOfO onevo vnovo amsfié fluovO —uovo @5090 NOQOO o¢0¢0 nQOOO #9000 ”0000 «veto ~09¢Q OGOOO hwevo owevo an0¢o 09000 ”@000 «@000 uncio onOQO NNOOO 06000 080'. 000- onw— Quo— buo— woo— hoo— Gao— mue— fluo- uno— Ono— o¢ou ato— uto— o¢0~ £00— ##0— 000- N00— ~00— ato— ome— wac— hac— eno— aflo— vac— mac— «@0— uno— Quo- 0N0— aflo- N~0— 9N0— aflo— GNO— 0N0— NNO- ~N0— ONO— 0~0— amo— N~0— 175 «ft N59 ‘flvu can Nb“— Oua— $00— Ono— 00'— 800 -Nfl~ N~N— «09 Oh" unfl— 0~0— boa— Duo— \Gva 0N0— 00— fig“— ~—N~ 000 008 ‘00—— \000 ‘9”0 08a nan uoeo n06 §8¢0 0Q! 009— 00N— tho— N90— can ha— \ONN \VOb ‘aco §oao huh—N §Ouufl . sh—~N swoon \_—o~ \ooou \—O¢— \uaoa ‘_00N \OQD— ‘vobu \vphfl \bbbfl s-oofl §aflON ‘toflu ‘cnflfl h—flflfl fiO—flfl ooo fla- sove— hono— bb—u 00~— one sou“ nau— , ton on» §uho um alum —:mm fimOfl uxda Elam IMAQ mum mxmfl cann v< —< and om¢ Dm< ¢m< 0m< uflmd o—m¢ Gum< bum< O—md Dumd Gum< a—md N—m4 ——m< Quad ~m< nQfid lm< dad Qm< A>An< 176 hun— NON— ”can «at— u!“— bou— ON!— N00— 00“. Ohm— '8'— ”0' Nb”— 00~. Goau ”am— when Qan— swan 0N0— Oh! N00— hau— ~0N- Qan— #o—u 0&0 ~0~— ‘aba— van 0~0— ab! Ovu— 0N- 00~— oun— Obn— ave nh—u ‘000— 000— 60— ONE N~0— ~N0 wan nuac keno— $000 Nb“ unn— amu— onu— «to Dan— hav— aha— afa— \Qfl— bun taan 0~0— NO? 080 Qua 0~0— haw v——— 0%“ «un— Gav 00~— \000 out fine" Qacfl unno— 080- urba— D—— qua a.” no»— to! \ono \—n@ uon unto nib uoauu baa teak fiOgh fi—oo unvo homon a—o— 0—0— .‘000— um: Emma omea Dmoa Ink: Gala 0949 cab—Q nah—fl ugh—Q uafihfl abnfl deo mmMU 1'77 Gob bun can 889 00a 80' «on 0~— ‘uua cow h¢ovu §QND 0‘»— «~— 0h" ONG— fitn— 0"— «v0— nua— (”an @0— 909 ch“ eav— oom— unv— §—ov Gon— nann— N89— w—v— bb—— «RN— '0— ‘Dca than won '88 mon— unfl— n0!— 0" man— NOD— "ab— @00— ahv 00¢ Nun 00¢ baa Nan ‘Oh _ me“ uh“ Nb" uofla a0— ‘00 $00 000— nut than— toma— aflo— awo— snoo— «bk ‘ooou £000 na— ‘00“— ‘—ON— fiat—— fine \—0 van who can" moa— ‘Na 000 “—0 ~90 one 00— 0~0— —0— N96— ‘O'fl— NNO~ 0~0— 0~0— ”—0— nO—A h0—A Quad 178 ‘Ohb «$0 vwa Nb—u 8&0 nQfl 6&0 0....— «O— to— on- he— onu— ov—u 000~ ‘Oflw N~0 00n— an uha Ono 000 005 onu— uO—u hD0~ ”bu— n—wu qua fibflw Ono— NON an— OD— DO—— 000— ‘00— Oman boa— OQG— ha“— 00“— ‘808 OD6 00“ Qua (on— Oak tun hi0 Nb— 050 On— Ou— Gnu Nua— ”0~— D00 bou— uno— ~0~— oo—~ awn— bo—u «aa— 000 ~06— 00"— 6'“— Own— hun— 00- 9~N— on“ DON 00»— ink Obo— ~00— ofl—~ 00~— Obe— hin— «wo flv__ 00N— Qa—q hfl—- o__~ when 60—— v.» 508 VON on“ can a—a— on“ fiVOfl ‘Nofl con can \—N0 000— Oak ska sea s¢fl uoa \fit so» unv nut \«0 sun ‘6‘ ‘aa ‘00“ fifiwfl ‘0— fivoau Q0- ”Q— ‘bna new a.» noo— o—o Ammo: mun: Hahn: gull mall «on: nun! hA A>A Oh>d find £5094 A>AQA Ended —44 179 NOO— \NOD— “wan «00~ 080— 000~ 8N0 000 «no ebb 800 who 90a 00' NC.— 000 0th boa 060 000 GNG nah baa— baa bah 0'— Dub 000 010 bra can ‘uflau hau— Goo O—b aa— 009— ~09— 099— 00”— van— boa— N09— «uh ”00 ”to Dh— v.9— bah Dav osc— unoa oni— 0&0 «N— haao— Goo 0~0 000 ope boo— can ebb 060 000 hav— ”00~ 00~ vou— 0~0 on» §aoa uao anon ‘flQQ ”00 fine. ”on 900 uaaa tona— uaov unno— no— 00.— ~00 fihoo 080 now— tuna §hoa ha! ‘000 coa— ~«h ha“ ‘080 yuan. ~au~ now who at! .hoau boa— avo n_o ao— _Qu— «oo— 0N0— ‘uvo ‘bo \—Q \QO hop nee ‘08 ‘0—0 \oou nah” uo—o \Oon \hhb ‘oaa ven— oa— one— to» ncao one $000 ‘0' O—a 999 fia— fibafl~ v—a ah." 0N0~ 005 0‘8 act QOOH Icom Nth Mmlh Beds «to oumé 180 NO!— wha— onu— 000~ 009— num— Cha— ban— «@0— QOQn 0Q!— 009— 009— 06?" ‘on§— £609" 009— ao—— @e—u too #00 00'" One— —D§— vou— 00~— N00 Obo— ava— fitn— ava— ava— tank 80!— con— 6'9— floo— ~0~— «flu— N8! ao- v—p \vva ~90 ”ha 000 00“— vah he'— 00— NOO— 00a— 09~— ‘00—— out cau— -b u—n con 00» ooh Nah 0~N— 00'— add— menu uoau obv— «Q— can. ave. ban. ‘a«—_ oeu— «o.— o_v nae- ___— won N~0 «on on» an» .95 v0!— aflo 09“— aka— ago man— 109»— nova vau— 0&6 shov— heav— unav— anv— suav— una— wov— nan— Goo— 00N— oeu— v—v Gua— Oh”— one— ‘uo utt— ana— N~0 ovflu Go.— out ‘bo ‘0" ‘Nfl not ‘na 50¢ ‘80 than— fiv— ~00— uco— oo— \QN rho ‘5— “—— bau— fi—o how.— twan— vtflu \an oeu— ‘56—— 000 uono ova— No— 00~— fi—Qb 08¢— ON!— #00— Hark A¢N ‘uoo 09¢ ml—fl> oou— av“ ‘0“ tax hank vac vfimQB ‘oub Ono nhmo3 soon eon ”hug? t—QO vbn nhmfit 00¢ fiv—G DON —0h huh 098 2923 ‘Ov- hfl— on u E vha won ‘Oaa OQXD @vv— Oflvu nav hbv vvv avv fiflev ohm 0b” 000 ova av” A>ALD bflv vow ‘N3 *2 4.5 show new «AD nba ‘00” n49 \—09 «an GAD «on ‘05” Dad: ‘68” On» an” bO~D Goa ‘bha ova IDQD ova noun haflb u—ov who new 00” «v» uva U—QD ovvu v—v— aov Nev Nov uov Gav \Oov can van aha new New ava vva 9v” baa mdnb «awn oaou Nflv- ano— APPENDIX E FORTRAN PROGRAM, PNP401.FT, CALCULATES ABSORBANCE AND THE FIRST DERIVATIVE OF ABSORBANCE This program is chained to from PNSF1.PA (Appendix D). Absorbance calculated absorbance for future and a link and the first derivative of absorbance are from intensity data passed from PNS?1.PA. The data is automatically stored on a magnetic disk use. The calculated values can then beplotted can be made to another program if desired. 182 183 Afl— . . a: . rash—Oh 4:: novon J 2:: Ann . .l— wag qua—h hO as. :35?— .q=._=:0®0— ._ VHF—:3 Ul—Pfimhx+P—.uha ulmsnhhx UB—bflm—X+gnomfi nlmA—u «man z\eflu‘=hl Ae.O—fl.. egg—my wand fizz. E 20 Eur—fisca— .a \.0.0—H.. egg—mo 92:3 5: Ed Hug—F.“ \.N— . . 641:..— .fiflm amba— .3Gm.3..r.¢<—.§.a.vu.g.a.—M 29.58 .Ol—Em E amp—Oak a :._flmzm.fll—gggm Ewan—ab 599—.— E .H>:.<>_¢Hn E muck—:4 mung—flog gm :4 .938!>MQF~> .n&: T..— u3+A Acu+n v3+ao+~ v3... A9: V3... an; v3+av+u vivllmgm :9: v3... 3:: v3... Ami; V3139: v3+au+n VBVIJVC vii—.mulm @OJu— 0D 8 mol~hfl.O—— v\.—.muaov> hmimufim .o*A—.— v3.7...” aO— v3+.nfiaov3+.flflafl v3+Ahv3nhm A333+.Nfiavv3+.afiaflv3+.vlaflv3+.a*: vivlufim V58IE~><¢ Eucml: “grader—fin 5?.— 95390440 H848 A120 av.m—H@VP§9& “00~ .nnu . a gram. 3 v3. 2 who AQaO— .vvflbug Mm:— E :0 <95“ 855—03 gnaw 93> gay—Gm". A — VIA—m A. ~§v\§§mlgm Vang—(D lbw—magma A ~ g» Eu 3 oh. our—32.9% Eng <+fl=mulbm <fl<+gmugm Run E EGO and nah—Om 8:503 HUS—g1 000— an $00— 0a 000 Quo- N000 O— UGO 186 b.h.¢3|fi.3fim.3.PJ—<~.MEQM.DM.vM.a—.NM.—M ZOE—8 .601: E mar—bug g Adz—EFF 23 Elk. flou— mfiflufig E mz—¢:OU any—E» .953: .503: awn—Ed.— E 8 gm»: “Ob vmmvmmvmm¢vh— mfiA.mnN~u~ on on Aamnuvrvmmn_ w¢9¢n mnmh m%—3 mFOAm mmOZ.efifiz¢Oh Amzmacev_._vmfim¢ ®©.®m_.®oA—In~.x~.ZHm—vh4m AAdU C I: ">— hhfiAZ—ZFIA_ohonx~ mna.mauu_ mm ca MDZ_PZOU _qumm— Nw.mm.omA~Izmm_vm— A—uo. emu-m3 .sz—om POAmuN .ZSOQ zwm EP_3 FOAmn~.VP¢Emom zmm_am@G—._Vndmm APOJmuvwmxd 4440 APOALHVPZHQ AA¢D quand OF mmmhm=